Egyptian Dental Journal (2006) 52, 1399-1407

Immunohistochemical Detection of Cytokeratin 14 in Developing of Rat Molars

Hisham I. Osman*+ and Azza S. Kora**

* Assistant professor of oral biology, faculty of dentistry, Alexandria University ** Lecturer of oral biology, faculty of dentistry, Alexandria University

Abstract Cytokeratins (CKs) are cytoskeletal intermediate filament proteins characteristic of odontogenic epithelia. The CK expression patterns of odontogenic epithelia are still poorly described. Most studies have searched for pools of CKs instead of individual polypeptides. The objective of this study was to clarify the immunohistochemical expression of individual CK polypeptides 14 in the enamel organ of rat molar at different developmental stages using monoclonal antibody LL002- Cks14.The results showed that all cells of the enamel organ were positive for CK14 and its configuration showed differences related to the stage-specific state of differentiation. A strong label for CK 14 was present at the inner dental epithelium at early bell stage, preameloblasts and amelobalsts It is conclude that the monoclonal antibodies to CK 14 are well documented and should serve as useful tools for tracing the development and differentiation of . The developing enamel organ may be a suitable model for investigating the relationship between keratin expression and cell function.

+Corresponding author. Tel.: +996502105253 E-mail address: [email protected]

Introduction Dental epithelial cells in cellular differentiation are not buds differentiate into various cell completely understood (1). types during development of tooth The cytoskeleton of the most germs, , cells is structured by scaffolding , stellate composed of actin microfilaments, reticulum, and outer enamel microtubules, and intermediate epithelium. The inner enamel filaments. Intermediate filaments, epithelial cells subsequently so named because their 10- differentiate into ameloblasts, nanometer diameter is intermediate which then form enamel. The between that of microfilaments mechanisms responsible for this (6 nanometers) and microtubules (23 nanometers), assemble into an

1 anastomosed network within the comprising the basic to neutral cytoplasm. They provide flexible cytokeratins 1-8, while the type I intracellular scaffolding whose group comprises the acidic function is to structure cytoplasm cytokeratins 9-20.. Thus, CK and to resist stresses externally polypeptides are markers for applied to the cell. Mutations that tissue-specific and stage-specific weaken this structural framework states of differentiation. increase the risk of cell rupture and Knowledge of CK expression cause a variety of human disorders patterns in dental epithelia is (2). useful not only for cell typing and The intermediate filaments are a monitoring the progression of large multigene family of fibrous epithelial cell differentiation, but protein that includes cytokeratins, also for understanding odontogenic which are characteristic of tumor histogenesis and epithelia and vimentin which is contributing to tumor classification typical of cells of mesenchymal (4). origin. The and The pattern of CK expression enamel organ are formed by within a particular epithelium epithelial cells which are varies with several parameters: cell characterized by the presence of type, developmental stage (5), state specific sets of cytokeratins (CK) of differentiation (6), anatomical (3). Cytokeratins are a subfamily of location (7), and degree of intermediate filament proteins and complexity (8). are characterized by remarkable biochemical diversity, represented Recent progress in understanding in epithelial tissues by at least 20 the biology of keratins, together different polypeptides. They range with the development of in molecular weight between 40 monoclonal antibodies to kDa and 68 kDa and isoelectric pH individual keratin proteins, (4.9 – 7.8). The individual provides the foundation for cytokeratin polypeptides are studying keratin expression in designated 1 to 20. Cytokeratin 1 normal and pathological oral has the highest molecular weight epithelia. The expression of CKs is and the highest isoelectric pH, closely linked to epithelium while cytokeratin 19 has the lowest differentiation, particularly during molecular weight and a low embryogenesis. They are useful as isoelectric pH. epithelial marker proteins because The cytokeratins are divided into of their high abundance, stability the type I and type II subgroups, and antigenicity. The pattern of the type II family members CK expression within a particular

2 epithelium varies with its helical regions separated by short anatomical location, non-helical linker sequences and developmental stage and state of flanked by non-helical globular differentiation. Epithelia can sequences commonly referred to as therefore be characterized by the head (N-terminal) and tail (C- specific pattern of polypeptides terminal) domains. Self-assembly they express (9). of CK14 leads to formation of intermediate filaments of 10 nm Different CKs have been shown diameter (15). to characterize epithelial type: In Only limited information is keratinized stratified epithelia, available on the cytokeratins of the such as the epidermis, CKs 1, 2, 5, enamel organ epithelium. 10, 11, and 14 are expressed, Immunohistochemical studies on whereas in nonkeratinized keratin expression in enamel stratified epithelia, such as the organs during development have oesophageal epithelium, CKs 4, 5, yielded rather variable and, in 13, 14 and 19 are found. The some cases, contradictory results relatively low molecular-weight (16, 17). Most studies have searched CKs (CKs 7, 8, 18, 19 and 20) for pools of CKs instead of have been identified in simple-type individual polypeptides. There is, (9) epithelia, including glandular , thus, a lack in the characterization (10,11) odontogenic and intestinal of individual CK expression in (12) tissues . Site-related differences dental epithelia. The aim of this have also been reported. CKs 2, 6, work was to analyze the and 16 characterize the keratinized distribution of individual CK stratified squamous epithelium of polypeptides 14 in odontogenic the hard palate. CK 13 is epithelia. Knowing that the tooth characteristic of the foreskin, development has been divided into whereas CK 15 marks the skin of three distinct phases; initiation, (13, 14). the sole morphogenesis and cell Cytokeratin 14, a member of the differentiation. family of acidic type I cytokeratins, consists of a conserved rod domain with four _-

3 Material and methods Ten normal, healthy, female omitted and goat antiserum pregnant rats were used in this antibody was substituted study .Developing mandibles were carefully dissected from the offsprings including lower first Staining Assessment molar regions buds from Cells were considered embryonic day 10 to postnatal day immunochemically positive for 7 so that to cover the period of anti cytokeratin 14 when distinct, embryonic and postnatal growth of reddish-brown, intracytoplasmic first molar development and were staining was identified. The fixed in 4% paraformaldehyde in staining intensity in cytokeratin phosphate-buffered saline. Tissues cases was categorized as follows: from postnatal rats were - = Negative, no staining reaction decalcified in 10% EDTA for + = slightly positive, densely about 3 weeks. Paraffin stained stained reaction sections H&E (5 um) were ++ = strongly positive, faintly prepared for routine histological stained cytoplasmic reaction examination. The tooth germs were classified into bud stage, cap The collected data were stage, bell stage and initial dental tabulated for evaluation of staining hard tissues formation. of cytokeratin at the different developmental stage of tooth formation

Immunohistochemistry An indirect streptavidin-biotin immunohistochemical technique (18) was used to detect the monoclonal mouse anti-human cytokeratin 14 (clone LL002)*. This monoclonal antibody reacts with the acidic intermediate filament protein (50kD) identified as cytokeratin 14 (CK14). Counterstaining was performed using Meyer's haematoxylin for one minute. For the negative controls the primary antibody was * DAKO, Copenhagen, Denmark

4 Results Positive reaction for monoclonal Early bell stage anti CK 14 in the stained sections During bell stage, cuspal among the epithelial cells of the morphogenesis continues and developing enamel organ as well as ameloblasts displayed a widely distributed undergo terminal differentiation. temporospatial pattern. Variations As at earlier stages, the distribution in staining intensity were verified of immunohistochemical staining among cells of different locations of CK 14 was temporally and during development of rat enamel spatially regulated. At this stage, organ shown in table 1. A closer Ck14 was located in the epithelial analysis of the CK14 expression in cells of the dental lamina and outer the various stages of epithelium. The inner development revealed the enamel epithelium revealed strong following: positively for CK 14. (fig.4). Remnants of the dental lamina showed an intense positive Initiation of tooth development reaction The and bud stage exhibited a weak immunoreactions At the early tooth formation, no for cytokeratin ( fig 5) immunoreactivity CK14 could be detected at the epithelial thickenings or in nearby tissues Late bell stage (fig.1). As histodifferentiation With terminal differentiation of proceeds, at early bud stage of the amelobalsts from the inner developing molar enamel organ, enamel epithelium, more extensive positive reactivity could only be staining of the enamel organ was seen in the stellate reticulum observed. There was intense (fig.2). None of the other cells of staining of the stratum the enamel organ were reacting intermedium (stronger than the positively at this stage of stellate reticulum) and weak or no development. No staining reaction of cytokeratin in immunoreactivity was detected in the inner enamel epithelium the ectomesenchymal tissues (fig.6). around the dental epithelium. At bud stage, the streak of staining cells extended to the epithelial bud Immunoreactivity for CK14 was (fig.3). observed among the progenitor cells of the cervical loop especially

5 within the outer and inner enamel layer and stratum intermedium epithelium (fig.7) exhibited a stronger positive reaction of CK14 in comparison to in developing the rest of the cell layer (fig.8, 9). molars After complete enamel formation, With the beginning of the the reduced enamel epithelium process of enamel formation, the showed a relatively fainted ck14 preameloblasts, cell staining (fig.10).

Fig.1 Molar tooth germ at bud stage stained with CK 14. No immunoreactivity of CK14 is detected either in all cell layers of the enamel organ or in the surrounding tissues X 200

Fig.2 Bud stage of tooth germ. Staining reaction extend from the oral surface epithelium to the central part of the budding dental epithelium (stellate reticulum) indicating a strongly positive reaction for CK14. The outer epithelial layer shows relatively weak staining X 400

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Fig.3 Further growth of the enamel organ. The enamel knot and the inner enamel epithelium showing marked immunoreactivity of CK14 X 400.

Fig.4 Early bell stage of molar tooth germ. All epithelial layers exhibited positive staining reaction. Note the strongly positive stain CK 14 of the inner enamel epithelium X 200.

Fig.5 Bell stage of tooth development ,weak Ck 14 reaction of the stellate reticulum

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Fig. 6 Late bell stage of molar tooth germ showing terminal differentiation of the ameloblasts with absence of staining reaction of the inner enamel epithelium. The stratum intermedium displays high expression of the CK14

Fig.7 Positive immunoreactivity of CK14 at the cervical loop of the developing enamel organ

Fig. 8 Positive reactivity of the ameloblast cell layer and stratum intermedium. Note the relatively staining intensity of the stellate reticulum and . X 200

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Fig. 9 Strong positive CK 14 staining of the ameloblasts X 400

Fig. 10 Uniform positive CK 14 of the reduced enamel epithelium. Remnants of dental lamina show weak immunoreactivity. Note the presence of positive cytokeratin staining in the covering epithelium indicating the presence of cytokeratin X 100

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Table 1: Expression pattern of CK14 among the developmental different stage of rat molar enamel organ

Enamel Organ Bud Ca Early bell Late bell Developing Tooth stage p stage stage sta ge Pre - Cervi IE St. OE IE St. OE Amelob RE St.I amelobl cal E R E E R E alsts E asts loop CK 14 + ++ ++ - + - ++ + + ++ ++ ++ +

- = Negative, + = slightly positive ++ = strongly positive IEE; Inner enamel epithelium, OEE; Outer enamel epithelium, St. R; stellate reticulum, St I; Stratum intermedium, REE; Reduced enamel epithelium

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Discussion organ during development Cells of the enamel organ observed in this study indicates a express keratin proteins typically temporo-spatial programme of associated with stratified keratin expression during epithelium. However, odontogenesis. In this work, one immunostaining patterns using type of cytokeratin was used specific antikeratin antibodies instated of using several types of change with development and cytokeratins and this provides a . These results more clear results about the demonstrate a relationship between temporarily changes that occur in keratin expression and the individual cell layers of the morphologic changes as enamel organ during development ameloblasts differentiate to This was in agreement with Leoset squamous cells of the reduced et al.(17) and they added that the use enamel epithelium. Changes in of monoclonal antibodies has keratin expression have been allowed detection of more changes previously documented in the in the reactivity of the stellate epidermis during fetal reticulum and stratum intermedium development and in normal than those seen with polyclonal differentiation of epidermal cells keratin antibodies. Cytokeratin 14 (19). In the present study, it may be (molecular weight 50 DK), an possible to relate changes in acidic (type I) cytokeratin protein, keratin staining with the dramatic is one of the cytokeratin pair changes in the enamel organ (50/58 KD) that distinguish ,ameloblast differentiation and stratified epithelial types from formation of reduced enamel simple epithelial types. It is a epithelium. homogenously expressed in all cells of the keratinizing squamous CK is a filament that plays a epithelium and is confined to the central role in epithelial tissue and, basal and parabasal cells in the like the polypeptides of nonkeratinizing squamous intermediate filaments in general, epithelium of the normal adult shows a high degree of tissue urinary tract (21) specificity. The pattern of CK distribution in the dental Cytokeratin 14 is an anchorage epithelium during tooth formation protein capable of preserving the is very complex (20). The mechanical integrity of epithelial differential reactivity of the cells (22). In the present work, the various cell-layers of the enamel immunohistochemical study

11 showed that the epithelial cells of was observed in the dental lamina, enamel organ and dental lamina in the reduced enamel epithelium expressed CKs 14 with slight and in almost all cells of the changes in their pattern during the enamel organ. Its absence in differentiation phase of regions of advanced amelogenesis odontogenesis. The most striking may suggest loss of CK14 as a finding was the presence of strong consequence of the cellular label for CK 14 in the inner secretory activity. enamel epithelium at early bell stage, which was changed into In the present study, during minimal amount indicated by weak enamel formation, the ameloblasts label at the late bell stage when the exhibited pronounced ameloblasts were fully accumulation of CK14. Similar differentiated. Domingues et al. (23), dynamics has earlier been were in agreement with the observed with E-cadherin, a cell previous result and stated that at adhesion molecule that is the early bell stage, the inner accumulated during the terminal enamel epithelium and the differentiation of ameloblasts (25). underlying mesenchymal cells This terminal differentiation of should be well connected to ameloblasts requires the integrity interact for developing of the cytoskeleton. Actin has been morphogenetic functions. Thus, shown to redistribute and the presence of CK 14 at this stage accumulate at the apical poles of might have a role in preserving and ameloblasts. E-cadherin has been supporting the epithelial- suggested to be involved in local mesenchymal interactions. On the accumulation of proteins during other hand, concurrently with polarization of epithelial cells dentin formation, the ameloblast to (26).Other molecules have also been be functional must be loosely involved in this process, such as connected to the mesenchyme to BMPs 4, 5 and 7 (27). It may be migrate and gain space for concluded that CK 14 could be depositing the enamel matrix. For regarded as a marker of ameloblast achieving this, it might be differentiation. In another study speculated that the ameloblast done by Tabata et al.,(9) they were needs to lose CK 14 to fully in accordance with previous disengage its anchorage. Crivelin conclusion and stated that CK14 et al. (24) supported the previous and amelogenin as ameloblast- statement and added that CK14 as differentiation markers, while the main intermediate filament of amelogenin is the postnatal odontogenic epithelium, which differentiation marker of

12 ameloblasts, the presence of CK14 provide the primary epithelial in ameloblasts 48–96 h earlier than attachment to enamel. At time of the expression of amelogenin tooth eruption, the reduced indicates that CK14 is a stage- ameloblast differentiates into a specific differentiation marker for typical squamous junctional ameloblasts. epithelial cell. Thus, the enamel organ is an unusual stratified The expression pattern of CKs epithelium which undergoes many 14 in early bell stage cells was functional changes during mostly similar to those of the late development, including functions bell stage, except for the staining typically associated with simple or of inner enamel epithelium. This glandular epithelia.(29-31) In this result was similar to those of study, a positively strong immune previous reports (28,16), except for clarified the formation of the REE the labelling of some individual by using staining the monoclonal CK polypeptides, such as CK 16 antibody cytokeratin as a marker and CK 17, the presence of which was behind the scope of this Several problems can limit the research. During differentiation of interpretation of antikeratin the inner enamel epithelium to immunohistochemical staining ameloblasts, a change of the cell data. (32) We used a highly polarity occurs. This previous sensitive staining method process is confirmed in this study (streptavidin-biotin), however the by the accumulation of strong extensive hard tissue positive staining of CK14 at both decalcification required in this ends of the ameloblasts. study produced weak keratin staining reaction. The ameloblast secretes enamel Conclusion matrix during the enamel This result demonstrates a apposition phase, and then relationship between keratin undergoes morphologic and expression and morphologic functional changes into a short, changes during different relatively cuboidal cell, the developmental stages of rat enamel reduced ameloblast. These cells organ and ameloblasts differentiate form part of the reduced enamel to squamous cells of the reduced epithelium (REE), a stratified enamel epithelium. Thus, the integument which protects enamel developing enamel organ may be a from the adjacent connective suitable model for investigating the tissue. During tooth eruption, the relationship between keratin REE fuses with oral epithelium to expression and cell function.

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اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ

آﺸﻒ هﺴﺘﻮآﻴﻤﻴﺎﺋﻰ ﻣﻨﺎﻋﻰ ﻋﻦ وﺟﻮد ﺳﻴﺘﻮآﺮاﺗﻴﻦ 14 ﻓﻰ ﺑﺮﻋﻢ اﻟﻀﺮوس ﻋﻨﺪ اﻟﻔﺌﺮان

إن اﻟﺴﻴﺘﻮآﻴﺮاﺗﻴﻦ ﻳﻌﺘﺒﺮ ﻣﻦ اﻷﻟﻴﺎف اﻟﺒﺮوﺗﻴﻨﻴﺔ اﻟﻬﻴﻜﻠﻴﺔ اﻟﻤﺘﻮﺳﻄﺔ اﻟﻄﻮل اﻟﻤﻤﻴﺰة ﻟﻠﺨﻼﻳﺎ اﻟﻄﻼﺋﻴﺔ اﻟﺴﻨﻴﺔ .. وﺣﺘﻰ اﻵن ﻳﻌﺘﺒﺮ وﺻﻒ اﻟﺘﺮآﻴﺐ اﻟﺒﻨﺎﺋﻰ ﻟﻬﺬﻩ اﻟﺨﻼﻳﺎ اﻟﺒﺮوﺗﻴ ﻨﻴﺔ اﻟﻤﺘﻮﺳﻄﺔ اﻟﻄﻮل ،ﻣﻦ اﻷﺷﻴﺎء اﻟﻘﻠﻴﻠﺔ ..وﻗﺪ ﺗ ﺮآﺰت ﻣﻌﻈﻢ اﻷﺑﺤﺎث اﻟﻌﻠﻤﻴﺔ ﺣﻮل اﻟﺴﻴﺘﻮآﻴﺮاﺗﻴﻦ ﻋﻠﻰ اﻟﺘﻌﺎﻣﻞ ﻣﻌﻪ آﻤﺠﻤﻮﻋﺔ آﺒﻴﺮة ﻣﺘﻜﺎﻣﻠﺔ ﻣﻦ اﻷﻧﻮاع اﻟﻤﺨﺘﻠﻔﺔ ﺑﺪﻻ ﻣﻦ اﻟﺒﺤﺚ ﺣﻮل ﻧﻮع ﻣﻌﻴﻦ ﻣﻦ اﻧﻮاﻋﻪ اﻟﻌﺪﻳﺪة .واﻟﻐﺮض ﻣﻦ هﺬا اﻟﺒﺤﺚ هﻮ اﻟﻜﺸﻒ ﻋﻦ ﻃﺮﻳﻘﺔ ﺗﻮزﻳﻊ اﻟﺒﺮوﺗﻴﻦ اﻟﺴﻴﺘﻮآﻴﺮاﻳﻦ رﻗﻢ 14 ﻓﻰ اﻷﻃﻮار اﻟﻤﺨﺘﻠﻔﺔ ﻟﺒﺮﻋﻢ اﻟﻀﺮوس ﻋﻨﺪ اﻟﻔﺌﺮان ﺑﺎﺳﺘﺨﺪام اﻟﻜﺸﻒ اﻟﻬﻴﺴﺘﻮآﻴﻤﻴﺎﺋﻰ اﻟﻤﻨﺎﻋﻰ .وﻗﺪ أﺳﻔﺮ اﻟﺒﺤﺚ ﻋﻦ إﻳﺠﺎﺑﻴﺔ ﺻﺒﻐﺔ اﻟﺴﻴﺘﻮآﻴﺮاﺗﻴﻦ 14 ﻟﺠﻤﻴﻊ اﻟﺨﻼاﻳﺎ اﻟﻤﻜﻮﻧﺔ ﻟﺒﺮﻋﻢ اﻟﻀﺮوس ،وان اﺧﺘﻠﻔﺖ درﺟﺔ اﺳﺘﺠﺎﺑﺘﻬﺎ ﺑﺎﺧﺘﻼف اﻟﻤﺮﺣﻠﺔ اﻟﺘﻄﻮرﻳﺔ اﻟﺘﻰ ﺗﻤﺮ ﺑﻬﺎ اﺛﻨﺎء ﺗﻜﻮﻳﻦ اﻟﺴﻨﺔ .وﻗﺪ أﻇﻬﺮ اﻟﺒﺤﺚ أﻳﻀﺎ وﺟﻮد ﺻﺒﻐﺔ اﻟﺴﻴﺘﻮآﻴﺮا ﺗﻴﻦ ﺑﺪرﺟﺔ اﻳﺠﺎﺑﻴﺔ ﻋﺎﻟﻴﺔ ﻓﻰ اﻟﻨﺴﻴﺞ اﻟﻄﻼﺋﻰ اﻟﺪاﺧﻠﻰ ﻟﺒﺮﻋﻢ اﻟﻀﺮوس ﻓﻰ ﻣﺮﺣﻠﺔ اﻟﺠﺮس اﻷوﻟﻰ واﻳﻀﺎ ﺧﻼل ﺗﻜﻮﻳﻦ ﻣﻴﻨﺎء اﻷﺳﻨﺎن . وﻧﺴﺘﺨﻠﺺ ﻣﻦ هﺬا اﻟﺒﺤﺚ إﻣﻜﺎﻧﻴﺔ اﻋﺘﺒﺎر ﺑﺮﻋﻢ اﻟﻀﺮوس آﻨﻤﻮذج ﻟﻠﺒﺤﺚ ﺣﻮل ﻋﻼﻗﺔ ﻇﻬﻮر اﻟﻜﻴﺮاﺗﻴﻦ ووﻇﻴﻔﺔ اﻟﺨﻠﻴﺔ .

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