Expression of fucosyltransferases in skin, conjunctiva, and cornea during human development

During human development, type-1-precursor, sialyl-Le a, and Le x antigens were present in the periderm of skin and eye at week 6. The Le x antigen disappeared from cornea at 10 weeks and then from skin at 20 weeks. H-type-1, Le a, Le b, sialyl-Le a, H-type-2, sialyl-Le x, and Le y were found in cornea, conjunctiva, and periderm between 10 and 20 weeks. They disappear from the skin (at week 20) and progressively reappear in skin derivatives, especially in the epithelium of sweat glands. The secretory part of the sweat gland is type-1-precursor and H-type-1 positive while its excretory part is Le a, Le b, sialyl-Le a, and Le y positive. On the eye surface the disappearance of Le x at 10 weeks and of the H-type-1, sialyl-Le x, and Le y at week 35 starts in the central cornea in front of the lens. The corneal epithelium and the conjunctiva have similar antigens to those of excretory and secretory parts of the sweat gland, respectively. Invaginations and folding of the epidermis might preserve the embryonic staining. We propose that fucosylation patterns are associated with the embryonic origin and differentiation stage of tissue. The early and transient presence of Le x is associated with FUT4 or FUT9 activities, while the late appearance of Lewis antigens is related to other alpha3-fucosyltransferases.     

The spectrum of cytokeratins expressed in the adult human cornea, limbus and perilimbal conjunctiva

The aim of this study was to detect a spectrum of cytokeratins (CK) present in the adult human cornea, limbus and perilimbal conjunctiva. Cryosections from seven corneo-scleral discs were fixed, and indirect immunofluorescent staining was performed using antibodies directed against CK1-CK10 and CK13-CK20. The percentage of positive cells was calculated in the epithelium of the cornea, limbus and perilimbal conjunctiva. Quantitative real time RT-PCR (qRT-PCR) was used to detect CK6 and CK18 expression in the corneal and conjunctival epithelium. The most intense staining present throughout the cornea was observed for CK3, CK5 and CK14; CK19 was found at the corneal periphery only. CK4 and CK10/13 revealed mild to moderate positivity mostly in the superficial layers of the cornea. The suprabasal cell layers of all examined areas showed a strong positivity for CK16. A heterogeneous staining pattern with a centrifugal decrease in the signal was observed for CK8 and CK18. CK5/6, CK14 and CK19 were present in the limbus, where a positive signal for CK3 was observed in the suprabasal and superficial cells only. In contrast to the cornea, CK15 appeared in the basal and suprabasal layers of the limbus. The perilimbal conjunctiva showed strong immunostaining for CK10/13, CK14 and CK19. A moderate signal for CK7 was detected in the superficial layers of the conjunctiva. qRT-PCR confirmed CK6 and CK18 expression in the corneal and conjunctival epithelium. The detailed characterization of the corneal, limbal and perilimbal conjunctival epithelium under normal circumstances may be useful for characterizing the changes occurring under pathological conditions.     

Observations on the morphology of the developing primate cornea: epithelium, its innervation and anterior stroma.

A survey is made of some ultrastructural features of the developing cornea of Macaca mulatta. The observations are confined to the anterior central area, starting with the lens vesicle stage and progressing through midgestation, when the morphologic characteristics of the cornea are fully established. Subepithelial filaments and some partially aggregated collagen fibrils are present in the earliest embryo and are of a size and appearance similar to those in the future vitreous cavity. Epithelial secretory activity points to, but does not prove direct contribution to the deposition of the acellular matrix components beneath it. No trace of a structured, orthogonal collagenous stroma can be visualized. The primitive endothelium forms prior to the fibroblast invasion of the distended filamentous matrix. Bowman's layer has undoubted epithelial contributions. Its aggregated collagen fibrils have approximately the same diameter as those of the anterior stroma. Intraepithelial appearance of single nerve fibers and fascicles takes place during the first trimester of gestation, as soon as the two continuous epithelial layers are formed. Terminal areas approach closely to the basal cell's nucleus, without touching it. The plasmalemma of the invaginating nerve fiber is surrounded by that of the epithelial cell in a mesaxon-like manner, with occasional gap junctions uniting adjoining neural and epithelial cell membranes. The fetal neurites contain microtubules, some clear vesicles and dense vacuoles resembling those of mature monamine and non-monamine neurons. Mitochondria are small and compact, their presence indicating a high rate of metabolic activity in the immature terminal area.     

Hypoxia as pathogenic factor affecting the eye tissues: The selective apoptotic damage of the conjunctiva and anterior epithelium of the cornea

The effect of acute hypoxia on the occurrence of apoptosis in eye cells in rats placed in a pressure chamber was studied. Selective primary lesion of cells of the conjunctiva and the anterior corneal epithelium was found. A possible role of the simulated hypoxic conditions in the dry eye syndrome pathogenesis, which is accompanied by primary lesion of cells in the anterior eye surface tissues is discussed.     

The roles of Pax6 in the cornea,retina, and olfactory epithelium of the developing mouse embryo

The roles of Pax6 were investigated in the murine eye and the olfactory epithelium by analysing gene expression and distribution of Pax6(-/-) cells in Pax6(+/+) <--> Pax6(-/-) chimeras. It was found that between embryonic days E10.5 and E16.5 Pax6 is autonomously required for cells to contribute fully not only to the corneal epithelium, where Pax6 is expressed at high levels, but also to the to the corneal stroma and endothelium, where the protein is detected at very low levels. Pax6(-/-) cells contributed only poorly to the neural retina, forming small clumps of cells that were normally restricted to the ganglion cell layer at E16.5. Pax6(-/-) cells in the retinal pigment epithelium could express Trp2, a component of the pigmentation pathway, at E14.5 and a small number went on to differentiate and produce pigment at E16.5. The segregation and near-exclusion of mutant cells from the nasal epithelium mirrored the behaviour of mutant cells in other developmental contexts, particularly the lens, suggesting that common primary defects may be responsible for diverse Pax6-related phenotypes.     

Proliferation, apoptosis and thymic involution.

The thymus reaches its maximal size at the age of 1 month in ICR mice and thereafter, the thymic cortex undergoes an exponential decline. This study was designed to compare the proliferation and apoptosis of thymocytes in different parts of the thymus of ICR female mice at the beginning and after the rapid phase of decline of the thymic cortical cellularity. The pattern of proliferation and apoptosis of the thymus was studied in situ in 1-month-old ICR female mice (10 mice) compared to mice at 7 months of age (10 mice). Staining for argyrophylic nucleolar organizer region by histochemistry was used to determine the proportion of type 2 thymocytes, which are considered as cells at S phase of the cell cycle. The mean number of type 2 cells in four random samples of 50 cells in each part of the thymus was defined as the proliferation index of this part of the thymus. In situ detection of apoptosis of thymocytes was carried out using the Apoptag kit, which can detect a single cell apoptosis. The mean number of apoptotic cells in five randomly selected fields of each part of the thymus was defined as the apoptotic index of this part of the thymus. The proliferation index of the peripheral cortex of the 1-month-old mice was 3.6 times higher than the proliferation index of the deep cortex and 5.8 times higher than the proliferation index of the medulla (P < 0.0001). The proliferation index of the peripheral cortex of the 7-month-old mice was reduced by 45% compared to the 1-month-old mice (P < 0.005). The apoptotic index of the corticomedullary junction of the 1-month-old mice was six times higher than the apoptotic index of the cortex and 18 times higher than the apoptotic index of the medulla. The apoptotic index of the thymic cortex was elevated by 66% in the 7-month-old mice compared to the 1-month-old mice (P < 0.0001). We conclude that there is a reduction of the proliferation index and an elevation of the apoptotic index of the thymic cortex in adult mice compared to young mice. These changes might account for the reduction of thymic cortical cellularity during thymic involution.     

Proteoglycan distribution in developing rabbit cornea

We used a staining procedure specific for sulfated glycosaminoglycans, cuprolinic blue dye (CBD), and immunohistochemical techniques to determine the histological distribution and ultrastructural organization of proteoglycans in developing rabbit cornea. We found several types of CBD-stained structures located throughout the corneal stroma, indicative of the distribution and perhaps the chemical heterogeneity of proteoglycans in this tissue. Keratan sulfate-specific immunohistochemical evidence supports our cytochemical findings. Our results suggest that low-sulfated keratan sulfate proteoglycans are found throughout most of the developing stroma, with the exception of the posterior margin of this tissue. Highly sulfated keratan sulfate proteoglycans in young fetal corneas, initially restricted to the subepithelial stroma, progressively extend to deeper portions of the stroma with development. Dermatan sulfate proteoglycans are located throughout the stroma, including the posterior margin. Invoking a recently published "oxygen-lack hypothesis" and correlating the tissue location of proteoglycans with the source of oxygen, we hypothesize that the distribution of proteoglycans in the developing rabbit cornea is related to the selective synthesis of keratan sulfate glycosaminoglycans under hypoxic conditions.     

Distribution and nature of desmosomes in the bovine developing ruminal epithelium.

Desmosomes entirely similar to those of the deeper layers of ruminal epithelium are seen in the luminal layers of the early fetal ruminal epithelium. In the older fetuses, these desmosomes have morphological features that reveal to some extent the occurrence of processes of keratinization. This could indicate that the basis for cell keratinization is already present in the fetus and that postnatal keratinization corresponds mainly to the full development of the prenatally existing pattern of differentiation.     

Early establishment of epithelial apoptosis in the developing human small intestine

In the adult small intestine, the dynamic renewal of the epithelium is characterized by a sequence of cell production in the crypts, cell maturation and cell migration to the tip of villi, where apoptosis is undertaken. Little is known about enterocytic apoptosis during development. In man, intestinal architectural features and functions are acquired largely by mid-gestation (18-20 wks); the question whether the establishment of enterocytic apoptotic processes parallels or not the acquisition of other intestinal functional features remains open. In the present study, we approached this question by examining enterocytic apoptosis during development of the human jejunum (9-20 wks gestation), using the ISEL (in situ terminal uridine deoxynucleotidyl nick-end labelling) method. Between 9 and 17 wks, apoptotic enterocytes were not evidenced. However, beginning at the 18 wks stage, ISEL-positive enterocytes were regularly observed at the tip of villi. Since the Bcl-2 family of proteins constitutes a critical checkpoint in apoptosis, acting upstream of the apoptotic machinery, we investigated the expression of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1). By immunofluorescence, we found that all homologs analyzed were expressed by enterocytes between 9 and 20 wks. However, Bcl-2 homologs underwent a gradual compartmentalization of epithelial expression along the maturing crypt-villus axis, to establish gradients of expression by 18-20 wks. Western blot analyses indicated that the expression levels of Bcl-2 homologs were modulated during morphogenesis of the crypt-villus axis, in parallel to their gradual compartmentalization of expression. Altogether, these data suggest that regulatory mechanisms of human enterocytic apoptosis become established by mid-gestation (18-20 wks) and coincide with the maturation of the crypt-villus axis of cell proliferation, differentiation and renewal.     

Cell cycle synchrony in the developing chicken lens epithelium.

Synchronous oscillations of DNA synthesis and histone 2B mRNA expression occur during normal development of 13- to 16-day-old embryonic chicken lens epithelium. At least four cycles were observed with peak values of DNA synthesis and histone 2B mRNA 5 to 10 times greater than baseline values. Fourier analysis of DNA synthesis identified a statistically significant oscillatory period of 18 hr, the approximate length of the cell cycle at this age. Minor components of 7-9 and 12 hr were also identified in the data sets. Lenses labeled with 3H-thymidine and analyzed by autoradiography at 13.8 days of embryogenesis revealed more than twice the number of labeled nuclei at this time than in lenses labeled 9 hr later; histone 2B mRNA followed this same pattern. These findings demonstrate that a significant population of cells is synchronized with respect to the cell cycle in the developing lens epithelium in ovo. The temporal pattern of mitosis may be the basis of the fiber cell architecture and consequent lens transparency.     

Apoptosis in the developing human heart resembles apoptosis in epithelial tissues

It is widely accepted that apoptosis plays an important role in the development of the heart as well as in different heart diseases. Despite extensive research efforts, many issues regarding apoptosis in the heart remain unsolved, including the detection of apoptotic cardiomyocytes, their morphological features, the mechanisms of their removal and the clinical significance of apoptosis in the heart. It has been suggested that fetal cardiomyocytes resemble epithelial tissues. To test this hypothesis, we analyzed the expression of an epithelial marker cytokeratin 18 (CK18) and caspase-cleaved-CK18, recognized by antibody M30, as well as the expression of cleaved caspase-3 and desmosomal and classical cadherins, major components of desmosomes and adherens junctions in fetal hearts in comparison to infant and adult human hearts. We found that, in fetal hearts, cardiomyocytes expressed CK18 and that apoptotic cardiomyocytes expressed caspase-cleaved CK18, being recognized by antibody M30. Furthermore, desmosomal and classical cadherins exhibited a membraneous reaction similar to epithelial tissues. In adults and children after the age of 6 months, cadherins were localized in the intercalated disks, cardiomyocytes lost CK18 expression and apoptotic cardiomyocytes were no longer recognized by M30. We conclude that apoptosis in the developing human heart resembles apoptosis in epithelial tissues, exhibiting different characteristics than in the adult human heart.     

Airway epithelium and apoptosis

Recent advances revealed that airway epithelium possesses versatile functions and plays a vital role in the mucosal defense and inflammatory responses. A maintenance of airway epithelium integrity is thus important and appears to be tightly regulated by a balanced cell proliferation and apoptosis. However, homeostasis of airway epithelium is likely affected by multiple environmental pathogens, irritants (reactive oxygen species, allergens, etc.), and toxins that may lead to various lung diseases. This review briefly summarizes airway epithelium apoptosis/proliferation in physiologic and pathological conditions along with various factors influencing airway epithelium homeostasis.