Effects of substrata and method of tissue dissociation on adhesion,cytoskeleton, and growth of chick retinal pigmented epithelium in vitro

Summary In this report we compare attachment, morphology, and growth of retinal pigmented epithelial (RPE) cells isolated by either EDTA or dispase digestion and plated onto either uncoated substrata (plastic or glass) or substrata derivatized by covalent conjugation of proteins of reconstituted basement membrane gel. We show that the derivatized substrata promote better initial attachment and subsequent cell growth than the uncoated substrata. These effects are independent of the method of dissociation of cells from the tissue. Cell morphology, however, is strongly affected by the method used for tissue dispersion. The dispase-dissociated cells are very flat, display a circumferential arrangement of microfilaments and elaborate extensive arrays of vinculin-containing cell-to-cell junctions. In contrast, EDTA-dissociated cells are much less spread, display straight microfilament bundles criss-crossing the cytoplasm and have less extensive cell-to-cell junctions. The protein-derivatized substrata also promote maintenance of differentiated traits such as pigmentation, by the RPE cells. Supported by Medical Research Council grant MA-9713 and by a grant from the R P Eye Research Foundation.     

Mechanics of spreading cells probed by atomic force microscopy

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.     

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-β1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.     

Adhesion of Human Glioma Cell Lines to Fibronectin, Laminin, Vitronectin and Collagen I Is Modulated by Gangliosides in vitro

Adhesion of eight cell lines, derived from human gliomas of different histological types, to fibronectin, collagen I, vitronectin, and laminin was investigated in vitro. The glioma cell lines were found to attach to these substrates to different extents. Interestingly, all cell lines strongly attached to laminin. In addition, glioma cell adhesion was found to be dose dependent. Moreover, adhesion of three cell lines to fibronectin and collagen I was partially inhibited and to vitronectin completely prevented by GRGDTP peptide, indicating the involvement of integrin receptors in glioma cell adhesion. We have demonstrated, recently, that gangliosides play an important role in promoting glioma cell invasion of the reconstituted basement membrane, Matrigel, in vitro. In order to study the mechanism of action of gangliosides in this process, the role of six gangliosides (GM1, GM3, GD3, GD1a, GD1b, and GT1b) in cell adhesion to the four proteins was investigated in three cell lines. Although all gangliosides, with the exception of GM3, were found to enhance cell adhesion to these proteins to different extents, GD3 proved to be the most effective adhesion-promoting ganglioside in all three cell lines. GM3 was found to inhibit cell adhesion to the four proteins in one cell line but enhanced cell adhesion in two other cell lines. The three cell lines were found to express both GD3 and gangliosides recognised by the A2B5 antibody. Furthermore, adhesion of the three cell lines to fibronectin, vitronectin, laminin, and collagen I was inhibited by incubation with A2B5, demonstrating the involvement of intrinsic cell membrane gangliosides in adhesion of glioma cells to these proteins. Taken together with the observation that gangliosides modulate integrin receptor function, these data suggest that gangliosides may play a central role in the control of the adhesive and invasive properties of human glioma cells.     

Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: A study of retinal regeneration in a novel animal model

Our research group has extensively studied retinal regeneration in adult Xenopus laevis. However, X. laevis does not represent a suitable model for multigenerational genetics and genomic approaches. Instead, Xenopus tropicalis is considered as the ideal model for these studies, although little is known about retinal regeneration in X. tropicalis. In the present study, we showed that a complete retina regenerates at approximately 30 days after whole retinal removal. The regenerating retina was derived from the stem/progenitor cells in the ciliary marginal zone (CMZ), indicating a novel mode of vertebrate retinal regeneration, which has not been previously reported. In a previous study, we showed that in X. laevis, retinal regeneration occurs primarily through the transdifferentiation of retinal pigmented epithelial (RPE) cells. RPE cells migrate to the retinal vascular membrane and reform a new epithelium, which then differentiates into the retina. In X. tropicalis, RPE cells also migrated to the vascular membrane, but transdifferentiation was not evident. Using two tissue culture models of RPE tissues, it was shown that in X. laevis RPE culture neuronal differentiation and reconstruction of the retinal three‐dimensional (3‐D) structure were clearly observed, while in X. tropicalis RPE culture neither ßIII tubulin‐positive cells nor 3‐D retinal structure were seen. These results indicate that the two Xenopus species are excellent models to clarify the cellular and molecular mechanisms of retinal regeneration, as these animals have contrasting modes of regeneration; one mode primarily involves RPE cells and the other mode involves stem/progenitor cells in the CMZ. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 739–756, 2014     

Effects of fibronectin-related peptides on cell spreading

Summary Four synthetic peptides related in amino acid sequence to the area of the fibronectin molecule thought to be involved in cell binding were tested for the ability to inhibit cell spreading promoted by several different attachment factors. Effects of the peptides were assayed on a number of cell types. For all cell types tested, peptides effective at inhibiting fibronectin-promoted cell spreading were found also to inhibit cell spreading promoted by human serum spreading factor (SF). These peptides were also capable of inhibiting to a lesser extent cell spreading promoted by other attachment factors, although this phenomenon was dependent on the cell type examined. This work was supported by National Institutes of Health grant CA-40475 Editor's statement This paper relates cellular attachment mediated by dissimilar molecules. As such it offers potential insight into common mechanisms of attachment. Gordon H. Sato     

Adhesion,proliferation, and differentiation of mesenchymal stem cells on RGD nanopatterns of varied nanospacings

The present report is an extension of our preceding publication in Biomaterials (2013) entitled “Effect of RGD nanospacing on differentiation of stem cells.” Cell-adhesive peptide arginine-glycine-aspartate (RGD) was nanopatterned on a non-fouling poly(ethylene glycol) (PEG) hydrogel, and mesenchymal stem cells (MSCs) derived from rat bone marrow were cultured on the patterned surfaces at nanospacings from 37 to 124 nm. Cell adhesion parameters such as spreading areas varied with RGD nanospacings significantly. The differences were well observed at both the first and eighth days, which confirmed the persistence of this nanospacing effect on our nanopatterns. The proliferation rate also varied with the nanospacings. Osteogenic and adipogenic inductions were undertaken, and a significant influence of RGD nanospacing on stem cell differentiation was found. The effect on differentiation cannot be simply interpreted by differences in cell adhesion and proliferation. We further calculated the fractions of single, coupled, and multiple cells on those nanopatterns, and ruled out the possibility that the extent of cell-cell contact determined the different differentiation fractions. Accordingly, we reinforced the idea that RGD nanospacing might directly influence stem cell differentiation.     

Type, location and role of glycosaminoglycans in cloned differentiated chick retinal pigmented epithelium

In clonal culture, colonies of 3–4 week old chick retinal pigmented epithelial cells exhibit Alcian Blue positive extracellular matrix (ECM) material on the surface of the cells. Alcian blue positive ECM is located between undifferentiated cells at the edges of the disc-shaped colonies and beneath the differentiated cells in the colony center. The latter material is associated with the basement membrane. The staining properties suggest that glycosaminoglycans (GAG) are present in these regions. Extraction of GAG from homogenates of colonies, followed by electrophoresis on cellulose acetate strips, results in three bands with mobilities similar to those of hyaluronic acid, heparan sulfate, and chondroitin sulfate, respectively. All three bands label with [³H]glucosamine, and the last two also label with [³⁵S]sulfate. The composition appeared to differ when colonies were grown in different media. Digestion of the GAG preparations with various enzymes suggests that bands II and III represent heparan sulfate and chondroitin sulfate, respectively, in colonies grown in Ham's F10g medium. The composition of band I is as yet undetermined. In minimal Eagle's medium (MEM), bands I and III consisted of hyaluronic acid and chondroitin sulfate, respectively, while band II had properties suggestive of a copolymer of heparan sulfate and an unidentified GAG. Cells release only one [³H]glucosamine-labelled GAG into the medium. This material has a mobility similar to hyaluronic acid and is digested by Streptomyces hyaluronidase, suggesting that it is hyaluronic acid. Staining with Alcian Blue at different pH suggests that it may represent the material associated with the upper surface of the cells. Some of the ECM located between the undifferentiated cells and associated with the basement membrane in the differentiated regions of the colonies stains with Alcian Blue at pH 1.0 and 0.2 suggesting that it may contain GAGs found in bands I and II. Colonies treated with medium containing 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of GAG synthesis, for 48 hr showed a reduced Alcian Blue staining of the ECM in the undifferentiated regions. After 72 hr of treatment with DON, the undifferentiated cells had detached from the plate, whereas the differentiated cells remained intact. The results suggest that the GAG may be involved in cellular adhesion, particularly of the undifferentiated cells.     

Location and possible function of fibronectin and laminin in clones of chick retinal pigmented epithelial cells

Summary Distribution and organization of the extracellular glycoproteins, fibronectin and laminin, in clonal cultures of chick retinal pigmented epithelial cells have been investigated using indirect immunofluorescence microscopy. Fibronectin is located on the apical and basal cell surfaces and between the cells in the undifferentiated regions of the colony (outer edge and stratified region). It seems to run parallel to intracellular microfilament bundles and to be associated with them across the cell membrane. In the differentiated region of thecolony (center), it is located exclusively on the basal cell surface and seems to be primarily associated with the collagen bundles of the basement membrane. The locations suggest that it may be necessary to stabilizing the sheet of differentiated cells in the colony center. In all regions except the outer edge of the colony, laminin is associated with the basal cell surfaces where it forms a meshwork of short, fine strands. The laminin has a totally different staining pattern from the fibronectin and does not seem to be associated with collagen bundles. The location suggests that laminin may be present in the basal lamina and may be involved in adhesion of the cells to the substratum. This work was supported by Medical Research Council of Canada (MA-6337).     

Upregulation of matrix metalloproteinase triggers transdifferentiation of retinal pigmented epithelial cells in Xenopus laevis: A Link between inflammatory response and regeneration

In adult Xenopus eyes, when the whole retina is removed, retinal pigmented epithelial (RPE) cells become activated to be retinal stem cells and regenerate the whole retina. In the present study, using a tissue culture model, it was examined whether upregulation of matrix metalloproteinases (Mmps) triggers retinal regeneration. Soon after retinal removal, Xmmp9 and Xmmp18 were strongly upregulated in the tissues of the RPE and the choroid. In the culture, Mmp expression in the RPE cells corresponded with their migration from the choroid. A potent MMP inhibitor, 1,10‐PNTL, suppressed RPE cell migration, proliferation, and formation of an epithelial structure in vitro. The mechanism involved in upregulation of Mmps was further investigated. After retinal removal, inflammatory cytokine genes, IL‐1β and TNF‐α , were upregulated both in vivo and in vitro. When the inflammation inhibitors dexamethasone or Withaferin A were applied in vitro, RPE cell migration was severely affected, suppressing transdifferentiation. These results demonstrate that Mmps play a pivotal role in retinal regeneration, and suggest that inflammatory cytokines trigger Mmp upregulation, indicating a direct link between the inflammatory reaction and retinal regeneration. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1086–1100, 2017     

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18–116 erg cm⁻². To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm⁻² for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm⁻². The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Y _s <50 erg cm⁻²), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (ΔF _adh) show that ΔF _adh decreases with increasingY _s , and equals zero around 45 erg cm⁻² for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.     

Characterization of Apin, a secreted protein highly expressed in tooth-associated epithelia

We previously reported expression of a protein by enamel organ (EO) cells in rat incisors, originally isolated from the amyloid of Pindborg odontogenic tumors called Apin. The aim of the present study was to further characterize the Apin gene and its protein in various species, assess tissue specificity, and clarify its localization within the EO. Northern blotting and RT-PCR revealed that expression of Apin was highest in the EO and gingiva, moderate in nasal and salivary glands, and lowest in the epididymis. The protein sequences deduced from the cloned cDNA for rat, mouse, pig, and human were aligned together with those obtained from four other mammal genomes. Apin is highly conserved in mammals but is absent in fish, birds, and amphibians. Comparative SDS-PAGE analyses of the protein obtained from bacteria, transfected cells, and extracted from EOs all indicated that Apin is post-translationally modified, a finding consistent with the presence of predicted sites for phosphorylation and O-linked glycosylation. In rodent incisors, Apin was detected only in the ameloblast layer of the EO, starting at post-secretory transition and extending throughout the maturation stage. Intense labeling was visible over the Golgi region as well as on the apices of ameloblasts abutting the enamel matrix. Apin was also immunodetected in epithelial cells of the gingiva which bind it to the tooth surface (junctional epithelium). The presence of Apin at cell-tooth interfaces suggests involvement in adhesive mechanisms active at these sites, but its presence among other epithelial tissues indicates Apin likely possesses broader physiological roles.     

Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins

Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the alpha(v)-containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism.     

Hepatocyte growth factor promotes the proliferation of human embryonic stem cell derived retinal pigment epithelial cells

Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC-RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC-RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA-665752, inhibited proliferation of hESC-RPE cells. However, differentiation of hESCs and human-induced pluripotent stem cells to a rostral fate and eye-field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC-RPE cells in human preclinical and clinical trials.     

Effects of hyperthermia on heat shock protein expression, alkaline phosphatase activity and proliferation in human osteosarcoma cells

Hyperthermia can be used as a possible adjuvant therapy in treatment of cancer patients. In this study, the direct effect of hyperthermia on osteosarcoma derived cell lines HOS85, MG-63 and SaOS-2 was investigated. Heat shock at 42 degrees C inhibited proliferation significantly in all three cell lines tested. Furthermore a sub-lethal heat shock (42 degrees C, 1 h) decreases alkaline phosphatase activity, the absolute marker for osteoblast-like cells, in all of the three cell lines. Hsp70 was expressed constitutively and was found to be upregulated in a time-dependent manner; by up to 150% in Western blot analysis. The results of this study indicate that heat shock has an inhibitory effect on human osteosarcoma cells. These data suggest that hyperthermia has an anti-tumour effect on cancers of the bone and might, therefore, become an adjuvant treatment option.     

Ovarian structure,folliculogenesis and oogenesis of the annual killifish Millerichthys robustus (Cyprinodontiformes: Cynolebiidae)

Cellular aspects of oocyte development of the Mexican rivulus Millerichthys robustus were morphologically described in order to analyze ovarian function and the cellular recruitment dynamics associating it with life history strategies of annual killifishes. Millerichthys is an iteroparous batch spawner with continuous oocyte recruitment and indeterminate fecundity with asynchronous development of the follicles. It has two ovaries of cystovarian type, with a central lumen, which communicates with the outside through the caudal region of the ovary, that is, the gonoduct. From the walls of the ovary, irregular lamellae composed of germinal epithelium and vascularized stroma project. Oogenesis starts with oogonial proliferation, found alone or in nests within the germinal epithelium. The oogonia come into meiosis becoming oocytes and advancing to the chromatin nucleolus stage and to early primary growth stage. Folliculogenesis is completed in the primary growth stage and cortical alveoli step. Follicles moves toward the stroma, but they continue to be attached to the germinal epithelium through the basement membrane until ovulation. The inclusion of fluid yolk in the follicles during the secondary growth stage was observed. During ovulation, the follicle collapsed, the oocyte was released into the lumen, and the constitutive elements of the post-ovulatory follicle complex remained in the stroma.     

Trichostatin A,a histone deacetylase inhibitor,suppresses proliferation and epithelial–mesenchymal transition in retinal pigment epithelium cells

The proliferation and epithelial–mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells are the major pathological changes in development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. Histone deacetylases (HDACs)‐mediated epigenetic mechanisms play important roles in controlling various physiological and pathological events. However, whether HDACs are involved in the regulation of proliferation and EMT in PRE cells remains unidentified. In this study, we evaluated the expression profile of HDAC family (18 genes) and found that some of class I and class II HDACs were up‐regulated in transforming growth factor‐β2 (TGF‐β2)/TGF‐β1‐stimulated RPE cells. Tricostatin A (TSA), a class I and II HDAC inhibitor, suppressed the proliferation of RPE cells by G1 phase cell cycle arrest through inhibition of cyclin/CDK/p‐Rb and induction of p21 and p27. In the meantime, TSA strongly prevented TGF‐β2–induced morphological changes and the up‐regulation of α‐SMA, collagen type I, collagen type IV, fibronectin, Snail and Slug. We also demonstrated that TSA affected not only the canonical Smad signalling pathway but also the non‐canonical TGF‐β/Akt, MAPK and ERK1/2 pathways. Finally, we found that the underlying mechanism of TSA affects EMT in RPE cells also through down‐regulating the Jagged/Notch signalling pathway. Therefore, this study may provide a new insight into the pathogenesis of PVR, and suggests that epigenetic treatment with HDAC inhibitors may have therapeutic value in the prevention and treatment of PVR.     

Effects of Th-2 type cytokines on human airway epithelial cells: interleukins-4, -5, and -13.

Proliferation and death of airway epithelial cells may be of importance in the pathogenesis of asthma. T-helper (Th)-2 response interleukins (IL)-4, -5, and -13 have the ability to induce proliferation of airway epithelial cells.     

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2‐Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4‐Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC‐pre‐treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF‐pre‐treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.     

Localization of DNA-synthesizing cells and cell proliferation pattern in developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens

Localization of DNA-synthesizing cells in the developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens was investigated. DNA-synthesizing cells were scattered throughout the proventricular epithelium during all developmental stages studied. The results indicate that there is no clear proliferative zone in the proventricular epithelium of the chicken. The labeling indices (LI) of proventricular epithelial cells were measured. On the 6.5th day of incubation, the LI of glandular epithelium reached 29.5 ± 1.5%. the highest value of all the stages studied. This extremely rapid cell proliferation can be considered to be a driving force for the elongation of the proventricular glands during the following stages. Just after hatching, the LI of both the glandular and luminal (non-glandular) epithelia significantly increased from those on the 18th day of incubation. It is suggested that the rise in LI possibly reflected proventricular growth to fit in the change in the method of nourishment after hatching. In 2 week old chickens, the LI of both the glandular and luminal epithelia were reduced to approximately 1%. The active production of embryonic chicken pepsinogen in all glandular epithelial cells of the embryonic chicken revealed that proliferation and differentiation are not necessarily exclusive during the embryonic stages of proventricular development.