Jack of all trades, master of each: the diversity of fibroblast growth factor signalling in eye development

A central question in development biology is how a limited set of signalling pathways can instruct unlimited diversity of multicellular organisms. In this review, we use three ocular tissues as models of increasing complexity to present the astounding versatility of fibroblast growth factor (FGF) signalling. In the lacrimal gland, we highlight the specificity of FGF signalling in a one-dimensional model of budding morphogenesis. In the lens, we showcase the dynamics of FGF signalling in altering functional outcomes in a two-dimensional space. In the retina, we present the prolific utilization of FGF signalling from three-dimensional development to homeostasis. These examples not only shed light on the cellular basis for the perfection and complexity of ocular development, but also serve as paradigms for the diversity of FGF signalling.

Artemisinin Inhibits the Migration and Invasion in Uveal Melanoma via Inhibition of the PI3K/AKT/mTOR Signaling Pathway

Uveal melanoma is the most common primary ocular neoplasm in adults, with many patients ending up developing liver metastasis and facing a significant reduction of their life expectancy due to the lack of efficient treatments. Artemisinin is an antimalarial drug that has been widely used in the clinic and whose anticancer properties have also been described. Its reported safety, affordability, and ability to reach the ocular tissues point that it has a potential therapeutic agent against uveal melanoma. In the present study, we found that a subantimalaria dosage of artemisinin significantly attenuated the migration and invasion potential of uveal melanoma cells, in a concentration-dependent manner. Assessment of the mechanisms underlying artemisinin anticancer action revealed that its use dramatically reduced the phosphorylation of PI3K, AKT, and mTOR in UM cells. Further inhibition of PI3K signaling, using LY294002, or of mTOR, by rapamycin, blocked the migration and invasion of UM cells similarly to artemisinin. In contrast, AKT or mTOR activator (Sc79 and MHY1485, respectively) attenuated the inhibitory effect of artemisinin on the migration and invasion abilities of UM cells, further validating that artemisinin’s anticancer effect is likely to be mediated via inhibition of the PI3K/AKT/mTOR pathway. Artemisinin also induced mitochondrial membrane potential loss and apoptosis of UM cells, having no significant toxic effect on normal retinal neuronal cells RGC-5 and epithelial cells D407. These findings and the reported safety of artemisinin’s clinical dosage strongly suggest the therapeutic potential of artemisinin in the prevention and treatment of uveal melanomas.

The Human Eye Transcriptome Atlas: A Searchable Comparative Transcriptome Database for Healthy and Diseased Human Eye Tissue

The applications of deep sequencing technologies in life science research and clinical diagnostics have increased rapidly over the last decade. Although fast algorithms for data processing exist, intuitive, portable solutions for data analysis are still rare. For this purpose, we developed a web-based transcriptome data-base, which provides a platform-independent, intuitive solution to easily explore and compare ocular gene expression of 100 diseased and healthy human tissue samples from 15 different tissue types collected at the Eye Center of the Universi-ty of Freiburg. To ensure comparability of expression between different tissues, reads were normalized across all 100 samples. Differentially expressed genes were calculated between each tissue type to determine tissue-specific genes. Unsupervised analysis of all 100 samples revealed an accurate clustering ac-cording to different tissue types. Cluster analysis based on known cell type-specific marker genes allowed differentiation of respective tissues. Several tis-sue-specific marker genes were identified. These genes were involved in tissue- or disease-specific processes, such as myelination for the optic nerve, visual perception for retina, keratinocyte differentiation for conjunctival carcinoma, as well as endothelial cell migration for choroidal neovascularization membranes. The results are accessible at the Human Eye Transcriptome Atlas website at https://www.eye-transcriptome.com. In summary, this searchable transcriptome database enables easy exploration of ocular gene expression in healthy and diseased human ocular tissues without bioinformatics expertise. Thus, it provides rapid access to detailed insights into the molecular mechanisms of various ocular tissues and diseases, as well as the rapid retrieval of potential new diagnostic and therapeutic targets.

Prenatal diagnosis of primary congenital glaucoma and histopathological features in a fetal globe with cytochrome p4501B1 mutations

Background/Objectives This study aims to report the developmental and histopathological features of ocular tissues from an electively aborted human fetus with mutations in cytochrome p4501B1, and thus predisposed to primary congenital glaucoma in comparison to an age-matched healthy fetal globe. Subjects/Methods Both eyes of two 17-week gestational aged fetuses, the first with CYP1B1 mutations and the second as healthy control fetus, were studied. Hematoxylin and eosin, Periodic acid–Schiff, Gomori’s trichrome, and Verhoeff–Van Gieson staining protocols in addition to immunohistochemistry staining using anti-cytochrome p4501B1, anti-fibrillin-1, and anti-4-hydroxy-2-nonenal antibodies, as primary antibodies, were performed to assess the effect of the mutations on tissue development, cytochrome p4501B1 protein expression, extracellular matrix structure, and oxidative stress in the developing fetus eye. Quantitative analyses were performed using ImageJ software. Student’s t-test was used for statistical analysis and P-values <0.05 were considered as significant. Results Delayed development in ocular tissues, decreased expression of cytochrome p4501B1 protein, irregular extracellular matrix structure, and increased oxidative stress biomarker were evident in the ocular tissues of the fetus with cytochrome p4501B1 mutations as compared to a normal globe from an age-matched fetus. Conclusion To the best of our knowledge, this is the first report of prenatal diagnosis of primary congenital glaucoma. We also describe histopathological changes in the primary congenital glaucoma-affected globes revealing the effect of cytochrome p4501B1 deficiency on ocular tissues during early fetal development contributing to the glaucoma phenotype.

Therapeutic Effects of Melatonin on Ocular Diseases: Knowledge Map and Perspective

Melatonin plays a critical role in the pathophysiological process including circadian rhythm, apoptosis, and oxidative stress. It can be synthesized in ocular tissues, and its receptors are also found in the eye, triggering more investigations concentrated on the role of melatonin in the eye. In the past decades, the protective and therapeutic potentials of melatonin for ocular diseases have been widely revealed in animal models. Herein, we construct a knowledge map of melatonin in treating ocular diseases through bibliometric analysis and review its current understanding and clinical evidence. The overall field could be divided into twelve topics through keywords co-occurrence analysis, in which the glaucoma, myopia, and retinal diseases were of greatest research interests according to the keywords burst detection. The existing clinical trials of melatonin in ocular diseases mainly focused on the glaucoma, and more research should be promoted, especially for various diseases and drug administration. We also discuss its bioavailability and further research topics including developing melatonin sensors for personalized medication, acting as stem cell therapy assistant drug, and consuming food-derived melatonin for facilitating its clinical transformation.

Nephronectin-Integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development

During development, cells aggregate at tissue boundaries to form normal tissue architecture of organs. However, how cells are segregated into tissue precursors remains largely unknown. Cornea development is a perfect example of this process whereby neural crest cells aggregate in the periocular region prior to their migration and differentiation into corneal cells. Our recent RNA-Seq analysis identified upregulation of Nephronectin (Npnt) transcripts during early stages of corneal development where its function has not been investigated. We found that Npnt mRNA and protein are expressed by various ocular tissues including the migratory periocular neural crest (pNC), which also express the integrin alpha 8 (Itgα8) receptor. Knockdown of either Npnt or Itgα8 attenuated cornea development, whereas overexpression of Npnt resulted in cornea thickening. Moreover, overexpression of Npnt variants lacking RGD binding sites did not affect corneal thickness. Neither the knockdown or augmentation of Npnt caused significant changes in cell proliferation, suggesting that Npnt directs pNC migration into the cornea. In vitro analyses showed that Npnt promotes pNC migration from explanted periocular mesenchyme, which requires Itgα8. Combined, these findings show that Npnt specifies and tunes cell migration into the presumptive cornea ECM by providing a substrate for Itgα8-positive pNC cells.

Viral S protein histochemistry reveals few potential SARS-CoV-2 entry sites in human ocular tissues

Despite the reported low expression of the primary SARS-CoV-2 receptor ACE2 in distinct ocular tissues, some clinical evidence suggests that SARS-CoV-2 can infect the eye. In this study, we explored potential entry sites for SARS-CoV-2 by viral S protein histochemistry on various ocular tissues and compared the staining patterns with RNA and protein expression of TMPRSS2 and ACE2. Potential viral entry sites were investigated by histochemistry using tagged recombinant viral S protein on 52 ocular tissue samples including specimens of the cornea, conjunctiva, lid margin, lacrimal gland tissue, retina, choroid, and RPE. In addition, ACE2 and TMPRSS2 immunohistochemistry were performed on the same ocular tissue, each with distinct antibodies binding to different epitopes. Lung tissue samples were used as positive controls. Finally, bulk RNA sequencing (RNA-Seq) was used to determine the expression of ACE2 and its auxiliary factors in the tissues mentioned above. S protein histochemistry revealed a positive staining in lung tissue but absent staining in the cornea, the conjunctiva, eye lid samples, the lacrimal glands, the retina and the optic nerve which was supported by hardly any immunoreactivity for ACE2 and TMPRSS2 and scarce ACE2 and TMPRSS2 RNA expression. Negligible staining with antibodies targeting ACE2 or TMPRSS2 was seen in the main and accessory lacrimal glands. In contrast, ocular staining (S protein, ACE2, TMPRSS2) was distinctly present in pigmented cells of the RPE and choroid, as well as in the ciliary body and the iris stroma. S protein histochemistry revealed hardly any SARS-CoV-2 entry sites in all ocular tissues examined. Similarly, no significant ACE2 or TMPRSS2 expression was found in extra- and intraocular tissue. While this study suggest a rather low risk of ocular infection with SARS-CoV-2, it should be noted, that potential viral entry sites may increase in response to inflammation or in certain disease states.

Analysis of BMP3 Variants In The Causality of Ocular Coloboma

Coloboma, a congenital disorder characterized by gaps in ocular tissues, is caused when the choroid fissure fails to close during embryonic development. Several loci have been associated with coloboma, but these represent less than 40% of those that are involved with this disease. Here, we describe a novel coloboma-causing locus, BMP3. Whole exome sequencing and Sanger sequencing of patients with coloboma identified three variants in BMP3, two of which are predicted to be disease causing. Consistent with this, bmp3 mutant zebrafish have aberrant fissure closure. bmp3 is expressed in the ventral head mesenchyme and regulates phosphorylated Smad3 in a population of cells adjacent to the choroid fissure. Furthermore, mutations in bmp3 sensitize embryos to Smad3 inhibitor treatment resulting in open choroid fissures. Micro CT scans and Alcian blue staining of zebrafish demonstrate that mutations in bmp3 cause midface hypoplasia, suggesting that bmp3 regulates cranial neural crest cells. Consistent with this, we see active Smad3 in a population of periocular neural crest cells, and bmp3 mutant zebrafish have reduced neural crest cells in the choroid fissure. Taken together, this data suggests that Bmp3 controls Smad3 phosphorylation in neural crest cells to regulate early craniofacial and ocular development.

Post-Mortem RT-PCR Assay for SARS-CoV-2 RNA in COVID-19 Patients’ Corneal Epithelium, Conjunctival and Nasopharyngeal Swabs

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease has been described to possibly be associated with ocular surface disturbances. However, whether the virus could invade ocular tissues still remains elusive. In the present study, we tried to investigate the post-mortem presence of SARS-CoV-2 RNA in corneal epithelium gathered by patients with an ante-mortem confirmed diagnosis of Coronavirus disease-19 (COVID-19). Cadavers with an ante-mortem confirmed diagnosis of moderate to severe COVID-19 were examined. Clinical and demographic features were retrieved from hospital patients’ notes. For each cadaver, corneal scrapings, conjunctival swabs (CS) and nasopharyngeal swabs (NPS) were collected to perform real-time reverse transcriptase polymerase chain reaction ((RT)-PCR) for SARS-CoV-2. Fourteen consecutive cadavers with an ante-mortem confirmed diagnosis of moderate to severe COVID-19 were examined. The last NPS performed ante-mortem confirmed SARS-CoV-2 infection in 12/14 (85.7%) patients. The mean death-to-swab time (DtS) was 3.15 ± 0.5 (2.10–5.1) h. The post-mortem NPS and CS found positive for SARS-CoV-2 RNA were 9/14 (64.3%) and 3/28 (10.7%), respectively. None of the corneal epithelium scrapes tested positive to RT-PCR for SARS-CoV-2 RNA. These data promote the SARS-CoV-2 as not able to contaminate the post-mortem corneal epithelium, while it can persist in different other structures of the ocular surface (i.e., the conjunctiva). It is reasonable to assume that such a contamination can occur ante-mortem too.