Early morphogenesis of ciliated cells in human oral cavity

Ciliated cells were found in the epithelium of the oral cavity of human embryos and fetuses starting from the seventh week of prenatal development. At the early stages of prenatal development (until the 13th week), cells with cilia cover most of the dorsal surface of the tongue and the soft palate, whereas they are found only near the gland ducts in the circumvallate and foliate lingual papillae after 17 weeks of development. The ultrastructure of the axoneme of cilia corresponds to the structure of motile cilia and is represented by nine microtubule doublets that surround the central pair of microtubule singlets. An immunohistochemical study performed on weeks 10–12 of development identified nerve endings associated with the ciliated cells. Until the 14th week of development, the cytoplasm of ciliated cells is immunopositive for NSE. The spatial distribution of ciliated cells in the tongue epithelium until the 13th week of development is not related to the morphogenesis of lingual papillae, and their role in the human oral cavity during the first trimester of pregnancy is unclear and requires further study.     

Innervation of developing human taste buds. An immunohistochemical study

 Morphological changes in developing human gustatory papillae during the 6th to the 23rd postovulatory week have been studied. The general innervation pattern of taste papillae and taste bud primordia was revealed immunohistochemically using antibodies against protein gene product 9.5 (PGP9.5), neurofilament H (NFH), neurofilament L (NFL), neurone-specific enolase (NSE), and tubulin. The autonomic and somatosensory nerve supply has been investigated using antibodies against substance P (SP), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), neuropeptide Y (NPY), the neuronal form of nitric oxide synthase (n-NOS), and, enzyme histochemically, NADPH-diaphorase. Nerve fibers approach the basal membrane of the lingual epithelium around the 7th postovulatory week and invade the epithelium of papilla-like structures at the 8th week, but some also penetrate the basal membrane of the non-papillary epithelium. They are in close contact with slender epithelial cells that are considered to be the taste bud’s progenitor cells. Early human taste buds situated at the anterior part of the tongue do not necessarily require a dermal (later fungiform) papilla. The NADPH-diaphorase reaction revealed positive results in dermal nerve fibers, but the immunohistochemical reaction against n-NOS was negative. Immunohistochemical detection of neuropeptides and vasoactive substances rendered negative results for developmental stages of 7–18 postovulatory weeks. By the 18th week, only SP was detected in dermal papillae, but not in the vicinity of taste buds’ primordia. Thus, autonomic and somatosensory nerves seem not to play a key role in formation and maintenance of early human taste buds. Accepted: 31 July 1997     

Optical and scanning electron microscopy observation of the mucosa of human fetal tongue

The structural features of the human foetal tongue have been studied in foetuses from 8th to 20th week of pregnancy. The characteristics of the developing papillae as well as of epithelial and mesenchymal layers have been pointed out. An early differentiation of the mesenchymal tissue has been observed, concerning phenomena of cellular condensation and reticular fibers organization both in superficial and deep layers. The hypothesis of the existence of straight interactions between epithelium and mesenchyme also in the developing human tongue mucosa has been suggested. Also the observations at SEM demonstrate that from the 8th to the 20th week the epithelial surface of the tongue reaches a stable structural pattern. From 11th week a characteristic cellular polymorphism occurs: cells with microvilli that diminish progressively, ciliated cells that disappear almost completely at the 20th week and cells whose free surface show microplicae, definitive stage of the tongue cell evolution.     

Ultrastructure of the human colon during ontogenesis

The ultrastructure of the colon of normal human embryos and fetuses was examined continuously from the 8th to 23rd week of pregnancy. The development of the colonic mucosa could therefore be presented at the moment, where the cellular differentiation nearly resembles those of adults. During the 8th week of pregnancy in the embryonic epithelium endocrine cells begin to differentiate. In the tunica submucosa unmyelinated axon bundles can already be found. The first goblet cells occur on 9 week old fetuses. The superficial epithelium carries a brush border covered by glycocalix. Osmiophile granules and enterochromaffin cells type 4 after Cristina are situated near the basal membrane. Underneath the tunica submucosa a thin layer of circular musculature has developed. From the 13th week onwards a stripe of longitudinal musculature joins the circular muscle layer in direction of the serosa. Between the muscle layers lie nerve bundles of the myenteric plexus. 14 or 15 week old fetuses show crypts. The endocrine cells can be classified into type 1, 2 and 4 after Cristina. In the 22nd week additionally to the lipid granules at the basal membrane, osmiophile bodies appear in the apical cytoplasm. At this stage a certain variety of intermediate forms between goblet- and endocrine cells occurs. Enterochromaffin cells type 3 after Cristina can be defined as well. A lamina muscularis mucosae has not yet arisen.     

A novel method to reconstruct epithelial tissue using high-purity keratinocyte lineage cells induced from human embryonic stem cells

The treatment of oral mucosa defect such as autologous oral mucosa caused by resection of oral mucosa carcinoma is still not ideal in clinical practice. However, Tissue engineering gives us the possibility to solve this problem. As we all know, Human embryonic stem cells (hESCs) have the ability to give rise to various cell types. We can take advantage of the totipotency of human embryonic stem cells to acquire keratinocytes. Directing the epithelial differentiation of hESCs can provide seed cells for the construction of epithelium tissue by tissue engineering. But, how to get high purity keratinocytes by induced stem cells then Applied to tissue engineering mucosa is an important challenge. We described a novel method to directly induce hESCs to differentiate into keratinocytes. Retinoic acid, ascorbic acid, and bone morphogenetic protein induced hESCs to differentiate into cells that highly expressed cytokeratin (CK)14. Our findings suggest that the retinoic acid, ascorbic acid and bone morphogenetic proteins induced hESCs to form high purity keratinocyte cell populations. In addition, we found that the highly pure keratinocyte populations reconstructed artificial tissue resembling epithelial tissue when inoculated in vitro on a biological scaffold.     

Scanning Electron Microscopical Studies of Developing Gustatory Papillae in Humans

Development and morphological changes of human gustatory papillaeduring postovulatory weeks 6–15 have been studied usingscanning and transmission electron microscopy. The first papillaof the tongue appears around postovulatory week 6 in its caudalmidline near the foramen caecum. In contrast, the dorsal epitheliumof the anterior part of the tongue shows only small hillock-or papilla-like elevations from week 6 on, which comprise anaggregation of 5–20 epithelial cells. From week 7 on,most prominent fungiform papillae develop near the median sulcusand at the margins of the anterior part of the tongue. At theirtops, the first primitive taste pores are found around week10; these are often covered with processes of adjacent epithelialcells. Most pores, however, develop around weeks 14–15.The maturation of taste buds does not coincide with the appearanceof taste pores, since taste bud cells are not fully differentiatedin the observed period of time. Fungiform papillae are developedbefore filiform papillae, which do not occur within the first15 weeks of gestation. Fungiform papillae tend to grow betweenweeks 8 and 15 of gestation, whereas the size of vallate papillaeseems to be constant during this period. Chem. Senses 22: 601–612,1997.     

Cytokeratin 14 is expressed in immature cells in rat taste buds

We analyzed the differentiation of taste bud cells, by precisely describing expression profiles of cytokeratins (CKs) 8 and 14 in relation to those of marker molecules including label of 5-bromo-2′-deoxy uridine (BrdU) injected. In rat circumvallate papillae, cell division was observed at the basal layer of the epithelium expressing CK14 and located outside taste buds. The progenitor cells began to migrate toward the apical surface and maintained CK14 expression at 1 day after BrdU injection (day 1). On the other hand, a minor population of newly divided cells was infrequently incorporated into taste buds and also maintained CK14 expression at day 1. In taste buds, the conversion of CK subtypes occurred from CK14 to cytokeratin 8 (CK8) at day 2–3, showing the differentiation from immature cells expressing CK14 into mature or maturing cells expressing CK8. Functionally matured cells such as taste receptor cells expressing inositol triphospate receptor type 3 (IP₃R3) never expressed CK14, suggesting that CK14 would be expressed only in immature cells. On the other hand, a small but distinct population of BrdU-positive cells still showed CK14 immunoreactivity in taste buds even at day 12, which might correspond to the cells that remain undifferentiated for a long period within taste buds.     

Emergence of TPA-resistant ‘satellite’ cells during muscle histogenesis of human limb

Human satellite cells, obtained by surgical biopsies of traumatized legs of healthy individuals, were grown in culture in the presence of different concentrations of the phorbol ester tetradecanoyl-phorbol 12 acetate (TPA). Satellite cells, after an initial duplicative period, fused into large multinucleated myotubes which readily synthesized myosin and acetylcholine receptor (AChR). The presence of TPA at concentrations up to 10(-7) M did not affect the differentiation pattern, while higher concentrations were toxic. Thus human satellite cells are capable of differentiating in the presence of phorbol esters which block differentiation of embryonic myoblasts [1]. We then examined the appearance of TPA-resistant cells during human muscle histogenesis, since we had observed that differentiation of human myoblasts from a 6-week-old limb was completely and reversibly inhibited by 10(-7) M TPA. Differentiation of myoblasts from 6-, 7- and 8-week-old fetuses was completely inhibited by TPA. Myoblasts from 10-week-old limbs did not form myotubes in the presence of TPA; however, immunohistochemical staining with an antimyosin antibody revealed the presence of a few mononucleated myosin-positive cells which escaped the TPA-induced block of differentiation. At 12 weeks of development, a few oligonucleated, myosin-positive myotubes developed in cultures treated with TPA, and the level of AChR expressed (measured as [125I] alpha-bungarotoxin bound) reached 20% of controls. At 14 weeks of development, about half of the cells in culture were TPA-resistant and by 16 weeks of development no major differences could be detected between control and treated cells. We conclude from these data that a population of TPA-resistant myogenic cells emerges between the 10th and 14th week of human limb development and suggest that this population represents satellite cells.     

Cytokeratin expression in human fetal tongue and buccal mucosa

Expression of cytokeratins (CK), a subset of intermediate filament (IF) proteins in epithelia, is developmentally regulated. CK expression may also change after malignant transformation. Our earlier studies on CK expression in human oral tumours and pre-cancerous lesions have shown specific changes in CK expression. We analysed CK expression in human tongue and buccal mucosa (BM) in fetuses in the embryonic age group of 16 to 27 weeks using biochemical and immunohistochemical techniques to find out whether there is any similarity in CK expression in human oral squamous cell carcinomas (SCC) and fetal oral tissues. CK 1, 8 and 18 were detected in a majority of samples using both techniques. Our earlier studies had shown aberrant expression of CK 1 and 18 in many of the oral SCC and leukoplakias. Studies by immunohistochemistry showed that these different CK antigens were expressed in different cell layers. CK 1(2) were present in the stratified epithelial layers whereas CK 8 and 18 were restricted to glandular epithelium. Till 27 weeks of gestation, both tongue and BM expressed CK 1, 8 and 18 along with CK 6 and 16. Thus, fetal tissues showed some similarities in CK pattern with their respective SCC.     

Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

Development of the taste buds in rat embryogenesis

Electron microscopic studies have been made on the developing taste buds in fungiform and vallate papillae of prenatal rats. Three stages of differentiation of these buds are described. The first stage is characterized by presence of the nervous fibers in the connective tissue of the papillae and dense granules of various size, as well as dense-cored vesicles (500-700 A in diameter) in the basal parts of some epithelial cells at the top of the papillae (16-17th days of gestation). The second stage is characterized by nerve processes entering the epithelium and by formation of afferent synaptic contacts between the differentiating epithelial cells and the nervous fibers (19th day of gestation). At the third stage, the cluster of differentiating epithelial cells attains a form which is similar to mature taste buds (21-22nd days of gestation). Thus, to the birthday of rats, differentiation of the basal parts of the taste buds takes place, whereas the apical parts of the taste buds remain undeveloped and do not communicate with the oral cavity. Peculiarities of fine structure of differentiating epithelial cells at the three stages are discussed.     

Ontogeny and distribution of certain endocrine cells in the human fetal large intestine

Summary In 9 fetuses, 9 to 24 weeks-old, the occurrence and relative distribution of argentaffin cells, as well as of cells immunoreactive to somatostatin (SRIF), glucagon-like polypeptide (GLI), pancreatic polypeptide (PP) and substance P (SP) were studied in five segments of the colon (appendix, cecum, ascending colon, descending colon, and rectosigmoid). For each colonic segment, data concerned with the occurrence of endocrine cells were expressed either as mean absolute numbers of specific cells per entire mucosal section, or as cell densities per mm³ of mucosa after calculation of the mucosal volume of the sections. Argentaffin, GLI, SRIF and PP immunoreactive cells are all present in relatively large numbers, scattered along the entire length of the colonic mucosa as early as the 9th–10th week of gestation, whereas substance P-containing cells occur sporadically and first appear during the 14th–17th week. Until the 20th week, with progressing embryonic development, an increase was determined in absolute numbers per section of all types of endocrine cells in all segments of the colon. This observation is clearly related to the general growth of the colonic mucosa, since cell densities per mm³ of mucosa do not greatly change or even decrease during gestation. However, it is possible that densities of argentaffin, GLI and BPP cells increase in the appendix around the 14th–17th week of gestation. Between the 20th and 24th week, absolute numbers of cells per section remain stable or slightly increase, while cell densities tend rather to decrease in all segments. These data demonstrate that some endocrine cells are present very early in the human fetal colon, but their functional significance remains to be elucidated.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM)     

Development of a highly reproducible three-dimensional organotypic model of the oral mucosa

In this report we describe the development of a standardized three-dimensional (3D) system of the human oral mucosa based on an immortalized human oral keratinocyte cell line (OKF6/TERT-2). The procedure takes approximately 2-3 weeks to complete and includes three main stages: preparation of collagen-embedded fibroblasts, addition of the mucosal component and airlifting of cultures to ensure adequate differentiation/stratification. This procedure results in a multilayer epithelial structure in which layers are organized similarly to the cells in native oral mucosa. Specifically, this model system consists of a stratum basale, having one layer of columnar to round cells, a relatively flattened stratum spinosum and stratum granulosum, and a non-keratinizing stratum corneum. This 3D system resembles the commercially available system based on the cell line TR146 (SkinEthic), with the exception that our model system does not contain dyskeratotic changes and has a submucosal component, and thus better represents the normal human mucosa and submucosa.     

Expression of cytokeratin-mRNAs in squamous-cell carcinoma and balloon-cell formation of human oesophageal epithelium

Summary Using digoxigenin-labelled cRNA probes, relationships between morphological characteristics and in situ hybridization for cytokeratin (CK)-mRNAs were analysed in cases of squamous-cell carcinoma of variable differentiation and in balloon-cell formation within the oesophageal mucosa. The present results were correlated to our previous findings on normal oesophageal epithelium. Our results from in situ hybridization study on oesophageal squamous-cell carcinoma provide strong evidence that changes in CK expression occur with differences in malignant potential. Cells of poorly differentiated carcinoma lose an ability to produce CK-mRNAs characteristic of their normal progenitor cells. Moderately differentiated and, still more pronounced, well differentiated carcinoma cells retain an ability to produce CKs characteristic of their tissue of origin (CK 6, CK 14, CK 15 and CK 19). Furthermore, well differentiated carcinoma cells may also gain an ability to synthesize new types of CKs that are not characteristic of the normal oesophageal epithelium (CK 8 and CK 18 characteristic of most simple epithelia, and CK 10 characteristic of keratinizing epithelia). Moreover, some oesophageal CK-genes are expressed in an obviously higher amount (CK 6, CK 14, and CK 19), but the expression of genes coding for the oesophageal differentiation-related CKs (CK 4 and CK 13) is obviously decreased or apparently lost. At the interface zone, observed in sections of well differentiated carcinomas, CK 8 and CK 18 mRNA were expressed in intermediate cell layers, and the centrally located cell layers were found positive for CK 10 mRNA. These findings largely extend the existing results from immunoblotting and immunohistochemical studies. The reduced or non-detectable expression of oesophageal differentiation-related CK-mRNAs (CK 4 and CK 13) on the appearance of balloon cells, suggests molecular changes that may be a marker for pathological progression. In addition, the abundant expression of CK 6 and CK 14 mRNA within areas of balloon-cell formation showing basal hyperplasia, and the higher expression of CK 19 in comparison with normal epithelium, points rather to de-differentiation than to normal vertical differentiation of the oesophageal epithelium. Whether CK-mRNAs can be used as biomarkers for evaluation of oesophageal pathologies remains to be further elucidated.     

Changes in enzyme activity in human trichinosis.

Changes in enzyme activity in human trichinosis have been studied. The activities of 19 enzymes and some isoenzymes were investigated in 12 patients from the 3rd to the 20th week. The activities of muscle-specific enzymes (CK, ALD and MK) were greatly increased with peaks during the 4th week; activities returned to normal ranges during the 7th and 8th weeks. Liver-specific enzymes (GTP, OCT, guanase, GLDH, SDH and MDH) and AP, ChE, cerulosplasmin remained within normal limits in all patients with the exception of a child who died. An explanation of the origin of hyperenzymemia is proposed on the basis of these investigations. The data obtained support the proposition that hyperenzymemia is of muscular origin and that the liver is seldom affected in case of trichinosis.     

The gastric mucosa of the human fetus. I. The surface epithelium

The ontogenesis of the surface epithelium in the gastric mucosa was studied by means of light and electron microscopy in 41 human foetuses ranging from 7th to 12th week of gestational age. The results obtained can be summarized as follows: 1) At the 7th week the gastric mucosa shows a simple pseudostratified epithelium; the epithelial cells are undifferentiated and filled, with glycogen clusters. 2) From the 8th week the epithelial surface shows small depressions that become deeper in the mesenchyme making the first bud of the gastric foveolae. 3) At the 9-10th week the gastric foveolae are more developed. The cells of the gastric epithelium can be therefore separated in two populations: a) the cells of the foveolae; b) the cell of the mucosal surfaces. 4) At the 12th week the cells of the mucosal surface become, on the basis of their histochemical and ultrastructural characteristics, surface mucous cells. The morphological differentiation is testified mainly by the transposition of the nuclei in the basal parts of the cells and by the gradual substitution of the cytoplasmic glycogen by mucous granules.     

Morphogenesis of the elastic fiber: an immunoelectronmicroscopy investigation

In this study a rabbit antiserum against human aortic elastin, which showed a high degree of species specificity in ELISA tests, was used to examine elastin fiber formation in the human fetal aorta between the ages of 14 and 23 weeks. Elastin was first detected by the antibody in the matrix of the 14-week-old specimen in association with the microfibrillar component. At this stage of development, the sections did not reveal structures morphologically identifiable as elastin. By the 17th week, discrete loci of elastin deposition were observed together with well-defined elastin fibrils. Only by the 23rd week did the aorta show the characteristic layering of elastic fibrils separating the myoblasts of the tunica media. In the latter specimen, the newly synthesized uncrosslinked elastin appeared to be unevenly distributed on the surface of elastin fibrils where it formed continuous strips of variable width arranged mostly in the form of spirals. This observation is discussed with respect to the proposals that the morphogenesis of elastic tissue is a dynamic process involving a close interrelationship between elastic fibrils and elastogenic cells and the morphogenetic movement of elastogenic cells plays an important role not only in the growth of elastic fibrils but also in the ultrastructural organization of the tissue.     

Early mother-infant relationships ofMacaca thibetana at Mt. Emei China

Quantitative data on early mother-infant relationships in the Tibetan macaque was collected during the first 23 weeks of infant life in spring, 1987, at Mt. Emei, China. During the first week of life, infants spent 98.3% of their time in ventroventral contact with their mothers. This contact rapidly decreased to 33.8% by the 4th week and thereafter to 0.85% by the 23rd week. Nipple contact decreased relatively slowly from 89.7% to 62.9% within the first 4 weeks of infant life and to 19.8% by the 23rd week. Ventrolateral and ventrodorsal contact appeared by the 2nd week, meanwhile, maternal restraining behavior appeared, and reached a peak by the 3rd week. The mother neither encouraged nor discouraged her infant's independence during 4–8th weeks. Maternal rejection of the infant was first observed when the infant was 11 weeks old and continued thereafter.     

Scanning electron microscopy of developing papillae on the tongue of human embryos and fetuses

The appearance and differentiation of papillae on dorsal andlateral surfaces of human embryonic and fetal tongues, at variousdevelopmental ages, were studied by scanning electron microscopy.Formaldehyde and phosphate buffer fixation provided satisfactorypreservation. At 8–9 weeks, the anterior two-thirds ofthe tongue showed no obvious signs of papillae. In contrast,just anterior to the sulcus terminalis rounded elevations wereseen, suggesting initial signs of circumvallate papillae. At10–13 weeks, the distribution and shape of elevationson the anterior two-thirds of the tongue indicated the beginningof fungiform papillae. Openings located on the dorsal surfaceof many of these fungiforms contained an amorphous central structureprojecting out of the papilla. First signs of foliate papillaewere seen at 10 weeks. At 15–18 weeks, fungiform and filiformpapillae were recognized, although sometimes their borders wereobscured by scaling epithelial cells. At 23–26 weeks,all papillae exhibited their adult form. ^*Presented, in part, at the VIth International Symposium onOlfaction and Taste, Gif-sur-Yvette, Paris, France, 15–17thJuly, 1977.     

Expression of cytokeratin 8, vimentin,syndecan-1 and Ki-67 during human tooth development

Spatio-temporal immunolocalizations of cytokeratin 8 (CK8), vimentin, syndecan-1 and Ki-67 were analyzed in ten human incisors and canine tooth germs between the 7th and 20th developmental weeks. CK8 expression was mild to moderate in the epithelial tooth parts, while it shifted from absent or mild in its mesenchymal parts, but few cells, sparsely distributed throughout the tooth germ, strongly expressed CK8. As development progressed, CK8 expression increased to strong in preameloblasts, while expression of vimentin increased to moderate in the epithelial and mesenchymal tooth parts, particularly in the dental papilla and sac. Co-expression of CK8 and vimentin was observed in some parts of the tooth germ, and was increasing in the differentiating preameloblasts and preodontoblasts. Syndecan-1 showed characteristic shift of expression from epithelial to mesenchymal tooth parts, being particularly strong in dental papilla, sac and cervical loops, while co-expression of Ki-67/syndecan-1 was strong in the dental papilla. Our study demonstrated spatio-temporal expression and restricted co-expression of the investigated markers, indicating participation of CK8 and vimentin in cell proliferation and migration, and differentiation of preodontoblasts and preameloblasts. Our data also suggest involvement of syndecan-1 in morphogenesis of the developing tooth crown and cervical loops, and together with CK8 and vimentin in differentiation of preameloblasts and preodontoblasts.