[Cell technologies as a basis for the development of regenerative principles for the treatment of lacrimal gland diseases]. / Kletochnye tekhnologii kak osnova razrabotki regeneratornykh printsipov lecheniya zabolevanii sleznoi zhelezy.

The lacrimal gland (LG) is a tubuloacinar exocrine gland composed of acinar, ductal, and myoepithelial cells. Three-dimensional distribution of acinar lobules, ducts, and myoepithelial cells is necessary for the effective functioning of the organ. LG is the main organ of immune surveillance of the ocular surface system. The embryogenesis of the gland is regulated by the interaction of genetic mechanisms, internal epigenetic (enzyme systems, hormones) and exogenous factors. There is no doubt that there is a clear genetic program for the implementation of the complex process of embryonic development. The mechanisms regulating LG organogenesis initiate the work of a huge number of structural oncogenes, transcription and growth factors, etc. Studying the expression and selective activity of regulatory genes during organ development, their participation in the differentiation of different cell types is a current trend at the nexus of clinical genetics, molecular biology, embryology and immunocytochemistry. Due to its relatively simple structure and accessibility, human LG is a suitable object for potential application in regenerative medicine. Development of a universal protocol for obtaining functional differentiated secretory epithelium of LG capable of expressing tissue-specific markers is an urgent task. Determining the nature and origin of stem cells and progenitor cells will allow the isolation and multiplication of these cells in culture. After obtaining a functionally active culture of LG cells, it is possible to create a model of autoimmune diseases.

Partial congenital arrhinia: never seen before adult presentation.

BACKGROUND: Arrhinia is defined as the partial or complete absence of the nasal structures. It is a defect of embryonal origin and can be seen in association with other craniofacial anomalies, central nervous system anomalies, absence of paranasal sinuses, and other palatal and ocular abnormalities. Very few patients with arrhinia have been reported so far in the history of modern medicine. CASE REPORT: This study reports an adult patient with congenital partial arrhinia and reviews the literature along with the embryological basis of such a rare disease. CONCLUSION: Arrhinia is a medical condition with scarce documentation in the literature. This article presents the clinical as well as radiological features of this rare entity.

Ectodysplasin-A signaling is a key integrator in the lacrimal gland-cornea feedback loop.

A lack of ectodysplasin-A (Eda) signaling leads to dry eye symptoms, which have so far only been associated with altered Meibomian glands. Here, we used loss-of-function (Eda-/-) mutant mice to unravel the impact of Eda signaling on lacrimal gland formation, maturation and subsequent physiological function. Our study demonstrates that Eda activity is dispensable during lacrimal gland embryonic development. However, using a transcriptomic approach, we show that the Eda pathway is necessary for proper cell terminal differentiation in lacrimal gland epithelium and correlated with modified expression of secreted factors commonly found in the tear film. Finally, we discovered that lacrimal glands present a bilateral reduction of Eda signaling activity in response to unilateral corneal injury. This observation hints towards a role for the Eda pathway in controlling the switch from basal to reflex tears, to support corneal wound healing. Collectively, our data suggest a crucial implication of Eda signaling in the cornea-lacrimal gland feedback loop, both in physiological and pathophysiological conditions. Our findings demonstrate that Eda downstream targets could help alleviate dry eye symptoms.

Punctal agenesis: Embryology, presentation, management modalities and outcomes.

Punctal agenesis is defined as the absence of the punctum occurring secondary to a failure of embryogenesis. This review synthesizes existing data on the embryology, anatomy, clinical presentation, symptomatology, management options and treatment outcomes of punctal agenesis. A foundational knowledge of the underlying embryologic and anatomical abnormalities is fundamental to understanding its clinical presentation and assists in choosing an appropriate management strategy. Existing outcomes data is generally favorable and suggests management with a step-wise approach can alleviate symptoms in patients across a spectrum of disease.

Preparation of Cells from Embryonic Organs for Single-Cell RNA Sequencing.

Although single-cell RNA sequencing (scRNA-seq) has become one of the most powerful methods available for transcriptome analysis, the quality of scRNA-seq data largely depends on cell preparation. Cell preparation from cultured cells and tissues requires different methods because of the inherent differences between these two categories of cells. Compared to cultured cells, tissues have more extracellular matrix, and the cells are generally more adherent and thus difficult to dissociate. The challenge is to achieve sufficient dissociation, cell counts, and viability all at the same time. This protocol describes approaches that help achieve these goals. These include a cold dissociation technique using cryophilic proteases active at cold temperature, timing of trituration during protease digestion, as well as filtration and washing methods that optimize cell viability and retention. Materials and equipment that optimize the process also discussed. © 2019 by John Wiley & Sons, Inc.

Molecular regulation of ocular gland development.

The tear film is produced by two ocular glands, the lacrimal glands, which produce the aqueous component of this film, and the meibomian glands, which secrete the lipidic component that is key to reduce evaporation of the watery film at the surface of the eye. Embryonic development of these exocrine glands has been mostly studied in mice, which also develop Harderian glands, a third type of ocular gland whose role is still not well understood. This review provides an update on the signalling pathways, transcription factors andextracellular matrix components that have been shown to play a role in ocular gland development.

Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland.

The tear-producing lacrimal gland is a tubular organ that protects and lubricates the ocular surface. The lacrimal gland possesses many features that make it an excellent model in which to investigate tubulogenesis, but the cell types and lineage relationships that drive lacrimal gland formation are unclear. Using single-cell sequencing and other molecular tools, we reveal novel cell identities and epithelial lineage dynamics that underlie lacrimal gland development. We show that the lacrimal gland from its earliest developmental stages is composed of multiple subpopulations of immune, epithelial and mesenchymal cell lineages. The epithelial lineage exhibits the most substantial cellular changes, transitioning through a series of unique transcriptional states to become terminally differentiated acinar, ductal and myoepithelial cells. Furthermore, lineage tracing in postnatal and adult glands provides the first direct evidence of unipotent KRT5+ epithelial cells in the lacrimal gland. Finally, we show conservation of developmental markers between the developing mouse and human lacrimal gland, supporting the use of mice to understand human development. Together, our data reveal crucial features of lacrimal gland development that have broad implications for understanding epithelial organogenesis.

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.

The lacrimal gland: development, wound repair and regeneration.

The lacrimal gland (LG) is important as it has a significant role in maintaining the stability of the microenvironment of the ocular surface. When a loss of function occurs in the LG, a significant reduction in tear production and dry eye disease (DED) may occur. A mammalian LG is a secretory gland consisting of acini and ducts. The interaction between epithelial cells and mesenchymal cells plays a major role during development and the self-restoration process of the gland. Some factors, such as fibroblast growth factor 10 and bone morphogenetic protein 7, are associated with these processes. Though several strategies for LG regeneration have been established, there is still a long way to go before there is clarity about LG stem cells. In this review, current knowledge on LG development, LG self-repair, DED and correlative regeneration therapies are summarized.

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.

FGF signaling activates a Sox9-Sox10 pathway for the formation and branching morphogenesis of mouse ocular glands.

Murine lacrimal, harderian and meibomian glands develop from the prospective conjunctival and eyelid epithelia and produce secretions that lubricate and protect the ocular surface. Sox9 expression localizes to the presumptive conjunctival epithelium as early as E11.5 and is detected in the lacrimal and harderian glands as they form. Conditional deletion showed that Sox9 is required for the development of the lacrimal and harderian glands and contributes to the formation of the meibomian glands. Sox9 regulates the expression of Sox10 to promote the formation of secretory acinar lobes in the lacrimal gland. Sox9 and FGF signaling were required for the expression of cartilage-associated extracellular matrix components during early stage lacrimal gland development. Fgfr2 deletion in the ocular surface epithelium reduced Sox9 and eliminated Sox10 expression. Sox9 deletion from the ectoderm did not affect Fgf10 expression in the adjacent mesenchyme or Fgfr2 expression in the epithelium, but appeared to reduce FGF signaling. Sox9 heterozygotes showed a haploinsufficient phenotype, in which the exorbital branch of the lacrimal gland was absent in most cases. However, enhancement of epithelial FGF signaling by expression of a constitutively active FGF receptor only partially rescued the lacrimal gland defects in Sox9 heterozygotes, suggesting a crucial role of Sox9, downstream of FGF signaling, in regulating lacrimal gland branching and differentiation.

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.

Transcription factors Runx1 to 3 are expressed in the lacrimal gland epithelium and are involved in regulation of gland morphogenesis and regeneration.

PURPOSE: Lacrimal gland (LG) morphogenesis and repair are regulated by a complex interplay of intrinsic factors (e.g., transcription factors) and extrinsic signals (e.g., soluble growth/signaling factors). Many of these interconnections remain poorly characterized. Runt-related (Runx) factors belong to a small family of heterodimeric transcription factors known to regulate lineage-specific proliferation and differentiation of stem cells. The purpose of this study was to define the expression pattern and the role of Runx proteins in LG development and regeneration. METHODS: Expression of epithelial-restricted transcription factors in murine LG was examined using immunostaining, qRT-PCR, and RT(2)Profiler PCR microarrays. The role of Runx transcription factors in LG morphogenesis was studied using siRNA and ex vivo LG cultures. Expression of Runx transcription factors during LG regeneration was assessed using in vivo model of LG regeneration. RESULTS: We found that Runx factors are expressed in the epithelial compartment of the LG; in particular, Runx1 was restricted to the epithelium with highest level of expression in ductal and centroacinar cells. Downregulation of Runx1 to 3 expression using Runx-specific siRNAs abolished LG growth and branching and our data suggest that Runx1, 2, and 3 are partially redundant in LG development. In siRNA-treated LG, reduction of branching correlated with reduction of epithelial proliferation, as well as expression of cyclin D1 and the putative epithelial progenitor cell marker cytokeratin-5. Runx1, Runx3, and cytokeratin-5 expression increased significantly in regenerating LG and there was modest increase in Runx2 expression during LG differentiation. CONCLUSIONS: Runx1 and 2 are new markers of the LG epithelial lineage and Runx factors are important for normal LG morphogenesis and regeneration.

The functional role of the Meis/Prep-binding elements in Pax6 locus during pancreas and eye development.

Pax6 is an essential transcription factor for lens, lacrimal gland and pancreas development. Previous transgenic analyses have identified several Pax6 regulatory elements, but their functional significance and binding factors remain largely unknown. In this study, we generated two genomic truncations to delete three elements that were previously shown to bind to the Meis/Prep family homeoproteins. One 3.1 kb deletion (Pax6(∆DP/∆DP)) removed two putative pancreatic enhancers and a previously identified ectodermal enhancer, while a 450 bp sub-deletion (Pax6(∆PE/∆PE)) eliminated only the promoter-proximal pancreatic enhancer. Immunohistochemistry and quantitative RT-PCR showed that the Pax6(∆PE/∆PE) pancreata had a significant decrease in Pax6, glucagon, and insulin expression, while no further reductions were observed in the Pax6(∆DP/∆DP) mice, indicating that only the 450 bp region is required for pancreatic development. In contrast, Pax6(∆DP/∆DP), but not Pax6(∆PE/∆PE) mice, developed stunted lacrimal gland and lens hypoplasia which was significantly more severe than that reported when only the ectodermal enhancer was deleted. This result suggested that the ectodermal enhancer must cooperate with its neighboring sequences to regulate the Pax6 ectodermal expression. Finally, we generated conditional knockouts of Prep1 in the lens and pancreas, but surprisingly, did not observe any developmental defects. Together, these results provide functional evidence for the independent and synergistic roles of the Pax6 upstream enhancers, and they suggest the potential redundancy of Meis/Prep protein in Pax6 regulation.

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.

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.

Prenatal diagnosis of bilateral dacryocystocele using 3-D/4-D ultrasound technology: a case report.

BACKGROUND: Bilateral dacryocystoceles develop as a result of obstruction of the nasolacrimal canal. The advances in obstetric sonography have led to increased diagnosis of this structural defect in the antenatal period as early as 24 weeks' gestation. CASE: We describe a case of bilateral dacryocystocele with clear visualization of this defect using 3-D/4-D sonography. CONCLUSION: Optimal visualization of this lesion using 3-D/4-D sonogram is helpful in providing better distinction of this lesion from other fetal facial lesions and can provide reassurance to anxious parents.

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.

Id2-, RORgammat-, and LTbetaR-independent initiation of lymphoid organogenesis in ocular immunity.

The eye is protected by the ocular immunosurveillance system. We show that tear duct-associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid-related orphan receptor gammat, lymphotoxin (LT) alpha1beta2-LTbetaR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity.

Eye formation in the absence of retina.

Eye development is a complex process that involves the formation of the retina and the lens, collectively called the eyeball, as well as the formation of auxiliary eye structures such as the eyelid, lacrimal gland, cornea and conjunctiva. The developmental requirements for the formation of each individual structure are only partially understood. We have shown previously that the homeobox-containing gene Rx is a key component in eye formation, as retinal structures do not develop and retina-specific gene expression is not observed in Rx-deficient mice. In addition, Rx-/- embryos do not develop any lens structure, despite the fact that Rx is not expressed in the lens. This demonstrates that during normal mammalian development, retina-specific gene expression is necessary for lens formation. In this paper we show that lens formation can be restored in Rx-deficient embryos experimentally, by the elimination of beta-catenin expression in the head surface ectoderm. This suggests that beta-catenin is involved in lens specification either through Wnt signaling or through its function in cell adhesion. In contrast to lens formation, we demonstrate that the development of auxiliary eye structures does not depend on retina-specific gene expression or retinal morphogenesis. These results point to the existence of two separate developmental processes involved in the formation of the eye and its associated structures. One involved in the formation of the eyeball and the second involved in the formation of the auxiliary eye structures.