Developmental change in the gene expression of transient receptor potential melastatin channel 3 (TRPM3) in murine lacrimal gland.

Transient receptor potential (TRP) channels are cation channels with ubiquitous expression. Various TRP channels are functionally active at the ocular surface and are involved in tear secretion and multiple inflammatory processes. So far, the impact of TRP channels regarding the development of the lacrimal gland (LG) is unclear. While investigating TRP channels in the LG, the TRPM3 channel presented itself as a promising candidate to play a role in the development and functioning of the LG. Therefore, Trpm3 expression was analyzed in different embryonic and postembryonic LGs. Thus, gene expression of TRPM channels including Trpm2, Trpm3, Trpm4 and Trpm6 was analyzed by quantitative RT-PCR in murine LGs at different developmental stages. Localization of TRPM3 in LGs was examined by immunohistochemistry. Primary LG epithelial cells (LGEC) and mesenchymal cells (MC) from newborn mice were cultured (either separately or collectively) for three days, and Trpm3 expression was analyzed in LGEC and MC. As a result, gene expression of Trpm2, Trpm4 and Trpm6 showed no significant difference in LGs in the different stages of development. However, Trpm3 gene expression was significantly higher in the embryonic stage than in the postnatal stage with the peak at E18. Postnatal, Trpm3 expression significantly decreased up to 28-fold until two years of age. Immunohistochemistry for TRPM3 revealed apical membranous expression in the excretory ducts, as well as in the acini of up to P7 old mice. Trpm3 expression in LGEC were significantly higher than that of MC. Our results indicate that Trpm3 expression in murine LG is age-dependent and peaks at age E18. Its expression is localized in the apical membrane of the glandular epithelium. However, its functional role still requires additional study in the LG.

FGF-induced Pea3 transcription factors program the genetic landscape for cell fate determination.

FGF signaling is a potent inducer of lacrimal gland development in the eye, capable of transforming the corneal epithelium into glandular tissues. Here, we show that genetic ablation of the Pea3 family of transcription factors not only disrupted the ductal elongation and branching of the lacrimal gland, but also biased the lacrimal gland epithelium toward an epidermal cell fate. Analysis of high-throughput gene expression and chromatin immunoprecipitation data revealed that the Pea3 genes directly control both the positive and negative feedback loops of FGF signaling. Importantly, Pea3 genes are also required to suppress aberrant Notch signaling which, if gone unchecked, can compromise lacrimal gland development by preventing the expression of both Sox and Six family genes. These results demonstrate that Pea3 genes are key FGF early response transcriptional factors, programing the genetic landscape for cell fate determination.

Alx4 relays sequential FGF signaling to induce lacrimal gland morphogenesis.

The sequential use of signaling pathways is essential for the guidance of pluripotent progenitors into diverse cell fates. Here, we show that Shp2 exclusively mediates FGF but not PDGF signaling in the neural crest to control lacrimal gland development. In addition to preventing p53-independent apoptosis and promoting the migration of Sox10-expressing neural crests, Shp2 is also required for expression of the homeodomain transcription factor Alx4, which directly controls Fgf10 expression in the periocular mesenchyme that is necessary for lacrimal gland induction. We show that Alx4 binds an Fgf10 intronic element conserved in terrestrial but not aquatic animals, underlying the evolutionary emergence of the lacrimal gland system in response to an airy environment. Inactivation of ALX4/Alx4 causes lacrimal gland aplasia in both human and mouse. These results reveal a key role of Alx4 in mediating FGF-Shp2-FGF signaling in the neural crest for lacrimal gland development.

miR-205 is a critical regulator of lacrimal gland development.

The tear film protects the terrestrial animal's ocular surface and the lacrimal gland provides important aqueous secretions necessary for its maintenance. Despite the importance of the lacrimal gland in ocular health, molecular aspects of its development remain poorly understood. We have identified a noncoding RNA (miR-205) as an important gene for lacrimal gland development. Mice lacking miR-205 fail to properly develop lacrimal glands, establishing this noncoding RNA as a key regulator of lacrimal gland development. Specifically, more than half of knockout lacrimal glands never initiated, suggesting a critical role of miR-205 at the earliest stages of lacrimal gland development. RNA-seq analysis uncovered several up-regulated miR-205 targets that may interfere with signaling to impair lacrimal gland initiation. Supporting this data, combinatorial epistatic deletion of Fgf10, the driver of lacrimal gland initiation, and miR-205 in mice exacerbates the lacrimal gland phenotype. We develop a molecular rheostat model where miR-205 modulates signaling pathways related to Fgf10 in order to regulate glandular development. These data show that a single microRNA is a key regulator for early lacrimal gland development in mice and highlights the important role of microRNAs during organogenesis.

Neuroprotective effects of food restriction on autonomic innervation of the lacrimal gland in the rat.

Inflammatory mechanisms and oxidative stress play important roles in age-related lacrimal gland (LG) degeneration as well as neural degeneration. Research suggests that caloric restriction can prevent age-related LG dysfunction and increase the life span of neurons. In the present study, we hypothesized that caloric restriction prevents age-related LG dysfunction by ameliorating the influence of inflammatory/oxidative stress on autonomic neurons controlling lacrimal function. We evaluated the effects of food restriction (FR) on inflammatory/oxidative status and on autonomic neural/neuroglial cell populations in LGs from aging rats. A total of 45 female albino rats were divided into young adult, aged, and aged-FR groups. The FR group was subjected to a 50% reduction in food from 14 to 20 months of age. LG samples were collected for each group and subjected to biochemical, histological, and immunohistochemical studies. LGs from aged-FR rats, rather than those from aged rats, showed preservation of their cellular structures, organelles, and Schwan cell units. LG preservation was associated with a marked decrease in inflammatory markers, an increase in cellular antioxidants, and the up-regulation of choline acetyltransverase, tyrosine hydroxylase, neuron-specific enolase and S100. These findings strongly suggest that in aged rats, both oxidative and inflammatory stressors directly contribute to LG dysfunction by mediating the degeneration of autonomic neurons, and that FR can protect against these effects.

Molecular Profiling of the Developing Lacrimal Gland Reveals Putative Role of Notch Signaling in Branching Morphogenesis.

Purpose: Although normal function of the lacrimal gland is essential for vision (and thus for human well-being), the lacrimal gland remains rather poorly understood at a molecular level. The purpose of this study was to identify new genes and signaling cascades involved in lacrimal gland development. Methods: To identify these genes, we used microarray analysis to compare the gene expression profiles of developing (embryonic) and adult lacrimal glands. Differential data were validated by quantitative RT-PCR, and several corresponding proteins were confirmed by immunohistochemistry and Western blot analysis. To evaluate the role of NOTCH signaling in lacrimal gland (LG) development, we used the NOTCH inhibitor DAPT and conditional Notch1 knockouts. Results: Our microarray data and an in silico reconstruction of cellular networks revealed significant changes in the expression patterns of genes from the NOTCH, WNT, TGFß, and Hedgehog pathways, all of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Our study also revealed new putative lacrimal gland stem cell/progenitor markers. We found that inhibiting Notch signaling both increases the average number of lacrimal gland lobules and reduces the size of each lobule. Conclusions: Our findings suggest that NOTCH-, WNT-, TGFß-, and Hedgehog-regulated EMT transition are critical mechanisms in lacrimal gland development and morphogenesis. Our data also supports the hypothesis that NOTCH signaling regulates branching morphogenesis in the developing lacrimal gland by suppressing cleft formation.

Regulating temporospatial dynamics of morphogen for structure formation of the lacrimal gland by chitosan biomaterials.

The lacrimal gland is an important organ responsible for regulating tear synthesis and secretion. The major work of lacrimal gland (LG) is to lubricate the ocular surface and maintain the health of eyes. Functional deterioration of the lacrimal gland happens because of aging, diseases, or therapeutic complications, but without effective treatments till now. The LG originates from the epithelium of ocular surface and develops by branching morphogenesis. To regenerate functional LGs, it is required to explore the way of recapitulating and facilitating the organ to establish the intricate and ramified structure. In this study, we proposed an approach using chitosan biomaterials to create a biomimetic environment beneficial to the branching structure formation of developing LG. The morphogenetic effect of chitosan was specific and optimized to promote LG branching. With chitosan, increase in temporal expression and local concentration of endogenous HGF-related molecules creates an environment around the emerging tip of LG epithelia. By efficiently enhancing downstream signaling of HGF pathways, the cellular activities and behaviors were activated to contribute to LG branching morphogenesis. The morphogenetic effect of chitosan was abolished by either ligand or receptor deprivation, or inhibition of downstream signaling transduction. Our results elucidated the underlying mechanism accounting for chitosan morphogenetic effects on LG, and also proposed promising approaches with chitosan to assist tissue structure formation of the LG.

Functional anatomy of the lacrimal gland in African black ostrich Struthio camelus domesticus in the embryonic and postnatal period.

The aim of the present study was morphological and histochemical analysis of the lacrimalgland (LG) in African black ostrich Struthio camelus domesticus in the embryonic and postnatalperiod. Studies were conducted on 50 ostriches aged between the 28th day of incubation until7 months old. Tissue sections were stained with haematoxylin and eosin, Azan trichrome,periodic acid-Schiff, Alcian blue pH 2.5, aldehyde fuchsin and Hale's dialysed iron. The LGin ostrich was classified as a tubulo-acinar type. The primordia of the lobes were determinedin the LG structure on the 28th day of incubation, whilst the weakly visible lobes with aciniand tubules were observed on the 40th day of incubation. Morphometric studies of the LGshowed steady growth, characterised by an increase in both length and width. Histometricmeasurements of lobe size showed little difference between the first, second and third agegroups, whilst in the fourth age group a marked increase in size of lobes was observed.The study showed that, apart from morphological changes, during the growth of the LGthe character of acid mucopolysaccharides changed. Sulphated acid mucopolysaccharideswere indicated, particularly with aldehyde fuchsin (AF) staining in the fourth age group.The Hale's dialysed iron (HDI) staining showed a low concentration of carboxylated acidmucopolysaccharides in the first and second age groups and a higher concentration in thethird and fourth age groups. Periodic acid-Schiff staining (PAS)-positive cells were observedin each age group, but only a small number of cells with a weakly PAS-positive reaction weredemonstrated in the first age group.

Manipulating the murine lacrimal gland.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

Age-related alterations in the lacrimal gland of adult albino rat: a light and electron microscopic study.

BACKGROUND: Age related changes in the lacrimal gland are associated with alterations in the structural organization and functional response in the gland of diverse mammalian species. Dry eye syndrome is one of the most common ocular problems in the world especially in old age. It results when the lacrimal gland fails to secrete proteins and fluid in sufficient quantity or appropriate composition. AIM OF THE WORK: The present study is designed to demonstrate the influence of aging on the structure of the lacrimal gland of albino rat and to provide a morphological basis to explain the pathogenesis of the dry eye syndrome with ageing. It also aims to carry out a comparative analysis of age-dependent changes in male and female rats and to address how the lacrimal gland ages in each sex. MATERIAL AND METHODS: Eighty albino rats were used in this study. The animals were divided into two age groups, young adult and senile. Tear secretion was measured using a modified Schirmer test. Corneal impression cytology of the anesthetized rats was done. The glands were subjected to gross morphologic examination, microscopic examination using H&E, PAS, Masson's trichrome and Giemsa stains. Electron microscopic examination was done in addition to quantitative histomorphometric estimations included acinar density, ductal count and mast cell count. RESULTS: Light microscopic examination of the lacimal glands of the senile rats revealed different pathological changes. These included acinar, ductal as well as stromal changes. Electron microscope examination of the lacrimal gland of the senile group showed a decrease in the electron dense secretory vesicles, mitochondrial swelling and lipofuscin-like inclusions were frequently seen in the cytoplasm of acinar cells in senile rats. CONCLUSION: The structural changes in the lacrimal glands of senile rats were associated with reduction in tear secretion as well as alterations in corneal epithelium. Gender difference in lacrimal gland structure was recorded.

Necessity of Smad4 for the normal development of the mouse lacrimal gland.

PURPOSE: Smad4, a key intracellular mediator in TGF-ß signaling, plays a critical role in the normal development of many tissues/organs. However, its functional role in the development of the lacrimal gland (LG) has never been reported. The aim of this study was to investigate the role Smad4 may play in the development of LG by using Smad4 conditional knockout mice and comparing their LG changes with the LG in normal control mice. METHODS: Smad4 in the LG, as well as in the lens, cornea, and ectoderm of the eyelids, was conditionally inactivated by using Pax6 promoter-driven Cre-transgenic mice. Lac Z reporter was used to visualize the developing LG by X-gal staining, and standard histologic approaches were used to reveal morphologic alterations. RESULTS: Inactivation of Smad4 resulted in reduction in the size and number of acini in the embryonic LG and in pigment accumulation within the LG, although it did not affect the formation of the primary lacrimal bud. After birth, the LG from Smad4-mutant mice developed slowly, and pigment was observed starting from the P7 mutant LG. Thereafter, the mutant LG was considerably smaller than the normal LG and was eventually replaced by adipose tissue. CONCLUSIONS: These results support the notion that Smad4 is essential for the normal development and maintenance of the mouse LG.

Barx2 and Fgf10 regulate ocular glands branching morphogenesis by controlling extracellular matrix remodeling.

The lacrimal gland (LG) develops through branching morphogenesis and produces secretions, including tears, that lubricate and protect the ocular surface. Despite the prevalence of LG disorders such as dry eye, relatively little is known about the regulation of LG development. In this study, we show that the homeobox transcription factor Barx2 is highly expressed in conjunctival epithelium, eyelids and ocular [lacrimal, harderian (HG), and meibomian (MG)] glands and is necessary for normal ocular gland and eyelid development. Barx2(-/-) mice show defective LG morphogenesis, absence of the HG, and defects in MG and eyelid fusion. Ex vivo antisense assays confirm the requirement for Barx2 in LG bud elongation and branching. Gene expression profiles reveal decreased expression of several adhesion and matrix remodeling molecules in Barx2(-/-) LGs. In culture, Barx2 regulates expression of matrix metalloproteinases (MMPs) and epithelial cell migration through the extracellular matrix. Fibroblast growth factors are crucial regulators of LG development and we show that Barx2 is required for Fgf10-induced LG bud elongation and that both Barx2 and Fgf10 cooperate in the regulation of MMPs. Together, these data suggest a mechanism for the effects of loss of Barx2 on ocular gland development. Intriguingly, salivary glands that also express a high level of Barx2 develop normally in Barx2(-/-) mice and do not show altered levels of MMPs. Thus, the function of Barx2 is specific to ocular gland development. Based on our data, we propose a functional network involving Barx2, Fgf10 and MMPs that plays an essential role in regulating branching morphogenesis of the ocular glands.

Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells.

Members of the Krüppel-like family of transcription factors regulate diverse developmental processes in various organs. Previously, we have demonstrated the role of Klf4 in the mouse ocular surface. Herein, we determined the role of the structurally related Klf5, using Klf5-conditional null (Klf5CN) mice derived by mating Klf5-LoxP and Le-Cre mice. Klf5 mRNA was detected as early as embryonic day 12 (E12) in the cornea, conjunctiva and eyelids, wherein its expression increased during development. Though the embryonic eye morphogenesis was unaltered in the Klf5CN mice, postnatal maturation was defective, resulting in smaller eyes with swollen eyelids that failed to separate properly. Klf5CN palpebral epidermis was hyperplastic with 7-9 layers of keratinocytes, compared with 2-3 in the wild type (WT). Klf5CN eyelid hair follicles and sebaceous glands were significantly enlarged, and the meibomian glands malformed. Klf5CN lacrimal glands displayed increased vasculature and large number of infiltrating cells. Klf5CN corneas were translucent, thicker with defective epithelial basement membrane and hypercellular stroma. Klf5CN conjunctiva lacked goblet cells, demonstrating that Klf5 is required for conjunctival goblet cell development. The number of Ki67-positive mitotic cells was more than doubled, consistent with the increased number of Klf5CN ocular surface epithelial cells. Co-ablation of Klf4 and Klf5 resulted in a more severe ocular surface phenotype compared with Klf4CN or Klf5CN, demonstrating that Klf4 and Klf5 share few if any, redundant functions. Thus, Klf5CN mice provide a useful model for investigating ocular surface pathologies involving meibomian gland dysfunction, blepharitis, corneal or conjunctival defects.

Lacrimal gland development and Fgf10-Fgfr2b signaling are controlled by 2-O- and 6-O-sulfated heparan sulfate.

Heparan sulfate, an extensively sulfated glycosaminoglycan abundant on cell surface proteoglycans, regulates intercellular signaling through its binding to various growth factors and receptors. In the lacrimal gland, branching morphogenesis depends on the interaction of heparan sulfate with Fgf10-Fgfr2b. To address if lacrimal gland development and FGF signaling depends on 2-O-sulfation of uronic acids and 6-O-sulfation of glucosamine residues, we genetically ablated heparan sulfate 2-O and 6-O sulfotransferases (Hs2st, Hs6st1, and Hs6st2) in developing lacrimal gland. Using a panel of phage display antibodies, we confirmed that these mutations disrupted 2-O and/or 6-O but not N-sulfation of heparan sulfate. The Hs6st mutants exhibited significant lacrimal gland hypoplasia and a strong genetic interaction with Fgf10, demonstrating the importance of heparan sulfate 6-O sulfation in lacrimal gland FGF signaling. Altering Hs2st caused a much less severe phenotype, but the Hs2st;Hs6st double mutants completely abolished lacrimal gland development, suggesting that both 2-O and 6-O sulfation of heparan sulfate contribute to FGF signaling. Combined Hs2st;Hs6st deficiency synergistically disrupted the formation of Fgf10-Fgfr2b-heparan sulfate complex on the cell surface and prevented lacrimal gland induction by Fgf10 in explant cultures. Importantly, the Hs2st;Hs6st double mutants abrogated FGF downstream ERK signaling. Therefore, Fgf10-Fgfr2b signaling during lacrimal gland development is sensitive to the content or arrangement of O-sulfate groups in heparan sulfate. To our knowledge, this is the first study to show that simultaneous deletion of Hs2st and Hs6st exhibits profound FGF signaling defects in mammalian development.

Nerve growth factor in the developing and adult lacrimal glands of rat with and without inherited retinitis pigmentosa.

PURPOSE: In the present study, we investigated lacrimal function and presence of the neurotrophin nerve growth factor (NGF) and its receptors in the lacrimal gland (LG) of normal rats and rats with inherited retinitis pigmentosa (IRP). MATERIALS AND METHODS: After anesthesia, modified Schirmer tests were performed on IRP rats and Sprague Dawley (SD) rats to measure tear function. LGs of developing and adult IRP and SD rats were removed and used for histological, immunohistochemical, and biochemical analyses. RESULTS: The results showed that basal tear secretion is reduced in IRP rats as compared with SD rats. NGF and NGF receptors are expressed in the LG of both rat strains. In SD rats, these NGF markers are low during early life and more elevated in adult life. Conversely in rats with IRP, NGF and its receptors decreased in adult life. CONCLUSIONS: The role of NGF in maintaining ocular surface integrity is well known. The observations of this study further support the hypothesis that neurotrophins play a role in modulating tear secretion and probably in preventing the deleterious effects of dry eye. This hypothesis is presented and discussed.

Sprouty2-modulated Kras signaling rescues Shp2 deficiency during lens and lacrimal gland development.

Shp2/Ptpn11 tyrosine phosphatase is a general regulator of the RTK pathways. By genetic ablation, we demonstrate that Shp2 is required for lacrimal gland budding, lens cell proliferation, survival and differentiation. Shp2 deletion disrupted ERK signaling and cell cycle regulation, which could be partially compensated by activated Kras signaling, confirming that Ras signaling was the main downstream target of Shp2 in lens and lacrimal gland development. We also showed that Sprouty2, a general suppressor of Ras signaling, was regulated by Shp2 positively at the transcriptional level and negatively at the post-translational level. Only in the absence of Sprouty2 could activated Kras signaling robustly rescue the lens proliferation and lacrimal-gland-budding defects in the Shp2 mutants. We propose that the dynamic regulation of Sprouty by Shp2 might be important not only for modulating Ras signaling in lens and lacrimal gland development, but also for RTK signaling in general.

Differential expression of espin isoforms during epithelial morphogenesis, stereociliogenesis and postnatal maturation in the developing inner ear.

The espins are a family of multifunctional actin cytoskeletal proteins. They are present in hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction. Here, we demonstrate that the different espin isoforms are expressed in complex spatiotemporal patterns during inner ear development. Espin 3 isoforms were prevalent in the epithelium of the otic pit, otocyst and membranous labyrinth as they underwent morphogenesis. This espin was down-regulated ahead of hair cell differentiation and during neuroblast delamination. Espin also accumulated in the epithelium of branchial clefts and pharyngeal pouches and during branching morphogenesis in other embryonic epithelial tissues, suggesting general roles for espins in epithelial morphogenesis. Espin reappeared later in inner ear development in differentiating hair cells. Its levels and compartmentalization to stereocilia increased during the formation and maturation of stereociliary bundles. Late in embryonic development, espin was also present in a tail-like process that emanated from the hair cell base. Increases in the levels of espin 1 and espin 4 isoforms correlated with stereocilium elongation and maturation in the vestibular system and cochlea, respectively. Our results suggest that the different espin isoforms play specific roles in actin cytoskeletal regulation during epithelial morphogenesis and hair cell differentiation.

Male NOD mouse external lacrimal glands exhibit profound changes in the exocytotic pathway early in postnatal development.

The lacrimal glands of male NOD mice exhibit many of the features of the human lacrimal gland in patients afflicted with the autoimmune disease, Sjögren's syndrome, including loss of secretory functions and lymphocytic infiltration into the lacrimal gland. To elucidate the early changes in the secretory pathway associated with development of Sjögren's syndrome, we investigated the organization of the exocytotic pathway in lacrimal glands of age-matched male BALB/c and NOD mice. Cryosections from lacrimal glands from 1 and 4 month male BALB/c and NOD mice were processed for confocal fluorescence and electron microscopic evaluation of different participants in exocytosis. No changes in apical actin filaments were noted in glands from NOD mice, but these glands exhibited thickening of basolateral actin relative to that seen in the BALB/c mice. Rab3D immunofluorescence associated with mature secretory vesicles was distributed abundantly in a continuous vesicular network concentrated beneath the apical plasma membrane in glands from 1 and 4 month BALB/c mice. In glands from 1 month NOD mice, rab3D immunofluorescence exhibited marked discontinuity and irregularity in the vesicular labeling pattern. While this change was also detected in glands from 4 month NOD mice, many of these glands exhibited an additional extension of rab3D labeling through the cell to the basolateral membrane. Electron microscopic analysis confirmed the formation of irregularly shaped, unusually large secretory vesicles in lacrimal glands from NOD mice. Quantitation of multiple secretory vesicles from electron micrographs revealed a significant (p< or =0.05) increase in the percentage of secretory vesicles incorporated into multivesicular aggregates in lacrimal glands from 1 and 4 month NOD mice compared to BALB/c mice. The M3 muscarinic receptor, a key signaling effector of exocytosis, was redistributed away from its normally basolateral locale in glands from BALB/c mice, with concomitant enrichment in intracellular aggregates in glands from NOD mice. These findings show that lacrimal glands in NOD mice as young as 1 month contain aberrant secretory vesicles with altered effector composition that undergo premature cytoplasmic fusion, and that changes in the distribution of the M3 muscarinic receptor occur within the same time frame.

The structure of the human semilunar plica at different stages of its development--a morphological and morphometric study.

In this study development of the semilunar plica was examined histologycally by making sections through the eyes of eleven foetuses at different stages of gestation, two newborns and an old man. We found that in the early stages of its development the semilunar fold covered a bigger part of the orbit and later did not keep up with the growth of the eyeball and the lids. In its development three different kinds of germinal glands could be seen in the semilunar plica. Beside poorly differentiated buds of the surface epithelium which can be classified as rudiments of the nictitating or Harderian gland, serous glands were detected which could be beginnings of Krause's glands. Additionally, a new kind of plica gland was identified in which ductular structures, with an onion skin appearance, could be discriminated from mucous acini. The surface of the plica developed slowly by an increase in the layers of its epithelium as well as by the maturation of the epithelial cells from a two-layered cuboidal to a multi-layered cylindrical epithelium. In general the palpebral side of the plica consisted of a higher number of epithelial layers and more goblet cells than its bulbar side. Moreover, the surface of the palpebral side appeared more irregular and enlarged by numerous pleatings of its epithelium. The initially loose mesenchymal connective tissue was soon condensed by firstly an increase of the number of cells and later by an increase in the fibre density. In the tight collagenous connective tissue no elastic or reticular fibres or cartilaginous structures could be found. Very dense vascularization of the plica semilunaris was seen early in development. The blood vessels rising from the root of the plica divided themselves into a central and a subepithelial vascular net. Later on, some of them showed an enlarged lumen and were covered by a thin layer of muscle cells. A few unmyelinated neurofibres were found next to blood vessels and glands. Muscle cells could not be detected. A very dense concentration of leukocytes in the plica even in early intrauterine development was very striking and could be observed in this kind for the first time. These were mostly lymphoplasmocytic elements but granulocytes and macrophages could also be seen around the blood vessels and in the subepithelial area where they were arranged as follicles. Some of these cells even passed through the epithelium and could be found in the conjunctival fissure. The discovery of dense infiltration with both specific and non-specific immune cells, abundant vascularization and secretory structures (goblet cells and surface enlargement) in the semilunar plica suggest that it plays an important role as a specialized organ in human eye protection. Its anatomical position at the medial border of the eye supports this theory. Its origin from the third eyelid of the mammals could be recognized to a certain extent.