The Tongue’s Ultrastructure Observation Of Epiplakin Knockout Mouse And Wild-type Mouse

Objective: Epiplakin(EPPK) belongs to the plakin family of cytolinkerproteins, which was indentified as an autoantigen in a serum specimenobtained from a patient with subepidermal blistering disease. EPPK isexpressed both in the stratified epithelium and in simple epithelium and isinvolved in interaction with intermediate filaments (IF). Under normalconditions, there were no detectable differences between wild-type andepiplakin “knockout” mice (EPPK-/-mice). However, during the closure ofwounds, EPPK-/-mice exhibited accelerated migration of keratin. Thekeratin filaments of EPPK-/-keratinocyte were thinner than those of normalkeratinocyte. Electron microscopy after immumostaining revealed that EPPKcolocalized with K5, K10and K6after wounding in wild-type mice. This dataindicates that EPPK accelerates keratin bunding in proliferating keratinocytesduring wound healing and suggest that EPPK might contribute toreinforcement of keratin networks under mechanical stress. Until now, nostudy about the tongue’s ultrastructure observations of EPPK-/-mice and wildtype mice has been reported. Laser scanning confocal microscopy revealedthat EPPK was expressed plentifully in the tongue of mice. Keratohyalinegranules are involved in the formation of keratin. To indentify the relation andfunction between EPPK and keratin, we observed and compared the lingualultrastructure of EPPK-/-mice and wild-type mice under transmission electronmicroscopy (TEM), especially keratohyaline granules, to further explore therelationship between EPPK and keratin and its function.Methods: The tongues of EPPK-/-mice and wild type mice were dividedinto8parts evenly and were numbered from tongue’s tip to tongue’s root.Firstly, the lingual structures of EPPK-/-mice and wild-type mouse wereobserved by light microscope. Then, the lingual ultrastructures of EPPK-/- mice and wild type mice were observed correspondingly in coronal plane byTEM.Results:1Light microscopic observations1.1The structure of lingual epithelium of wild-type mouse can be dividedinto four parts. The cells of the basal layer were cubic lied like wave. Thecells of the spinous layer were more flat than the cells of basal layer. Theobservation by light microscopy revealed that keratohyaline granules were notfound in both the basal layer and the spinous layer. The cells of the granularlayer were flat. There were plentiful keratohyaline granules, which were notof uniform size （see Fig.1、3、5）, distributing layer upon layer over thegranular layer. Keratohyaline granules were strong basophilic in HE staining.The cells of the horny layer, where the keratohyaline granule was not found,were more flat than those of the spinous layer. The keratohyaline granulesdistributed in a linear array in the free part of the lingual papillae and were lessthan those of the part between papillae and papillae (see Fig.1、3、5). Therewas no papilla in the root of the lingual epithelium where four layers wereobserved. The horny layer was thinner than the tip of the tongue and there wasno significant difference between the root of the tongue and the tip of thetongue in the other three layers. The keratohyaline granules of the root of thetongue were less than the tip of the tongue (see Fig.7).1.2The lingual epithelium of EPPK-/-mouse can be divided in four layers.The basal cells like a cylinder and arrayed like wave. The cells of the spinouslayer were round. There was no keratohyaline granules found in both the basallayer and the spinous layer. The keratohyaline granules scattered in thegranular layer were few and the distribution was not in regular pattern (see Fig.2、4、6). No keratohyaline granules was found in the horny layer. The base ofthe lingual papillae like pillar and no keratohyaline granule found here. Fewkeratohyaline granules were found in the free part of the tongue (see Fig.2、4、6). No papillae were found in the root of the tongue, and the structure of theepithelium here was no different from the part between papillae and papillae from the tip of the tongue. The only difference we observed was thekeratohyaline granules, which scattered in the root of the tongue were lessthan those of the tip of the tongue (see Fig.8).2TEM observations2.1At low magnification （×4000）, we observed that the lingualepithelium of the wild-type mouse was regular. The basal cells were ovalshaped, and the major axis was perpendicular to the base. The major axis ofthe cells of the spinous layer, where few round keratohyaline granules couldbe found, was parallel to the base. Many round keratohyaline granules werefound in granular layer, especially the part between papillae and papillae (seeFig.9). No keratohyaline granule was found in the horny layer. The basal cellsof lingual epithelium from the EPPK-/-mouse were pillar shaped. Nokeratohyaline granule was found in the basal layer. The cells of the spinouslayer were round and almost no keratohyaline granule was found here. Fewround keratohyaline granules scattered in the granular layer （see Fig.10）. Noabnormal cell junctions were found in every layers of lingual epithelium.2.2At high magnification （×25000）, the electron density of peripheralportion of keratohyaline granules from wild-type mouse was higher than thecentral site of keratohyaline granules （see Fig12、14）. The cell junction wasinseparable (see Fig12、14、16). The tonofilaments were normal. The electrondensity of keratohyaline granules from EPPK-/-mouse was uniform（seeFig.13、15）. The cell junctions were separable（see Fig.13、15、17）.Mitochondrions and the Golgi apparatus in the cytoplasm were not foundunusual significantly. The tonofilaments of EPPK-/-mouse were thinner thanthose of wild-type mouse using NIH imaging software to analyze electronmicrographs.Conclusion:1These observations suggested that the keratohyaline granules from thelingual epithelium of EPPK-/-mouse were less than those of the lingualepithelium of wild-type mouse significantly by light microscope.2These observations suggested that the keratohyaline granules from the lingual epithelium of EPPK-/-mouse was smaller than those from the lingualepithelium of wild-type mouse, and was lack of the high electron density edge.In addition, the cell junctions were separable and the tonofilaments of EPPK-/-mouse were thinner.In a word, the defection of epiplakin gene leaded to ultrastructure changesof the lingual epithelium, especially the keratohyaline granules.