Expression of Nephronectin in the Descemet's membrane of mouse corneas during development and adult homeostasis.

Nephronectin (Npnt) is an extracellular matrix (ECM) protein with pleiotropic functions during organogenesis, disease, and homeostasis. Although the ECM plays a crucial role during development and homeostasis of the adult cornea, little is known about the expression of Npnt in the mammalian cornea. Here, we investigated the expression of Npnt during early embryonic and postnatal development, and in adult mouse corneas. We combined ultrastructural and immunohistochemical analyses to study the early formation of the Descemet's membrane and how the expression of Npnt relates to key basement membrane proteins. Our section in situ hybridization and immunohistochemical analyses revealed that Npnt mRNA is expressed by the nascent corneal endothelial cells at embryonic day (E) 14.5, whereas the protein is localized in the adjacent extracellular matrix. These expression patterns were maintained in the corneal endothelium and Descemet's membrane throughout development and in adult corneas. Ultrastructural analysis revealed discontinuous electron dense regions of protein aggregates at E18.5 that was separated from the endothelial layer by an electron lucent space. At birth (postnatal day, P0), the Descemet's membrane was a single layer, which continuously thickened throughout P4, P8, P10, and P14. Npnt was localized to the Descemet's membrane by E18.5 and overlapped with Collagens IV and VIII, Laminin, and Perlecan. However, the proteins subsequently shifted and formed distinct layers in the adult cornea, whereby Npnt localized between two Collagen VIII bands and anterior to Collagen IV but overlapped with Laminin and Perlecan. Combined, our results reveal the expression of Npnt in the mouse cornea and define its spatiotemporal localization relative to key basement membrane proteins during the formation of the Descemet's membrane and in the adult cornea. Understanding the spatiotemporal expression of Npnt is important for future studies to elucidate its function in the mammalian cornea.

Visualization of RNA virus infection in a marine protist with a universal biomarker.

Half of the marine virosphere is hypothesized to be RNA viruses (kingdom Orthornavirae) that infect abundant micro-eukaryotic hosts (e.g. protists). To test this, quantitative approaches that broadly track infections in situ are needed. Here, we describe a technique-dsRNA-Immunofluorescence (dsRIF)-that uses a double-stranded RNA (dsRNA) targeting monoclonal antibody to assess host infection status based on the presence of dsRNA, a replicative intermediate of all Orthornavirae infections. We show that the dinoflagellate Heterocapsa circularisquama produces dsRIF signal ~ 1000 times above background autofluorescence when infected by the + ssRNA virus HcRNAV. dsRNA-positive virocells were detected across > 50% of the 48-h infection cycle and accumulated to represent at least 63% of the population. Photosynthetic and chromosomal integrity remained intact during peak replication, indicating HcRNAV infection does not interrupt these processes. This work validates the use of dsRIF on marine RNA viruses and their hosts, setting the stage for quantitative environmental applications that will accelerate understanding of virus-driven ecosystem impacts.

BMP3 inhibits TGFß2-mediated myofibroblast differentiation during wound healing of the embryonic cornea.

Often acute damage to the cornea initiates drastic tissue remodeling, resulting in fibrotic scarring that disrupts light transmission and precedes vision impairment. Very little is known about the factors that can mitigate fibrosis and promote scar-free cornea wound healing. We previously described transient myofibroblast differentiation during non-fibrotic repair in an embryonic cornea injury model. Here, we sought to elucidate the mechanistic regulation of myofibroblast differentiation during embryonic cornea wound healing. We found that alpha-smooth muscle actin (αSMA)-positive myofibroblasts are superficial and their presence inversely correlates with wound closure. Expression of TGFß2 and nuclear localization of pSMAD2 were elevated during myofibroblast induction. BMP3 and BMP7 were localized in the corneal epithelium and corresponded with pSMAD1/5/8 activation and absence of myofibroblasts in the healing stroma. In vitro analyses with corneal fibroblasts revealed that BMP3 inhibits the persistence of TGFß2-induced myofibroblasts by promoting disassembly of focal adhesions and αSMA fibers. This was confirmed by the expression of vinculin and pFAK. Together, these data highlight a mechanism to inhibit myofibroblast persistence during cornea wound repair.

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas.

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

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 nor 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, focal adhesion kinase, and Rho kinase. Combined, these data suggest that Npnt augments cell migration into the presumptive cornea extracellular matrix by functioning as a substrate for Itgα8-positive pNC cells.

Recapitulation of normal collagen architecture in embryonic wounded corneas.

Wound healing is characterized by cell and extracellular matrix changes mediating cell migration, fibrosis, remodeling and regeneration. We previously demonstrated that chick fetal wound healing shows a regenerative phenotype regarding the cellular and molecular organization of the cornea. However, the chick corneal stromal structure is remarkably complex in the collagen fiber/lamellar organization, involving branching and anastomosing of collagen bundles. It is unknown whether the chick fetal wound healing is capable of recapitulating this developmentally regulated organization pattern. The purpose of this study was to examine the three-dimensional collagen architecture of wounded embryonic corneas, whilst identifying temporal and spatial changes in collagen organization during wound healing. Linear corneal wounds that traversed the epithelial layer, Bowman´s layer, and anterior stroma were generated in chick corneas on embryonic day 7. Irregular thin collagen fibers are present in the wounded cornea during the early phases of wound healing. As wound healing progresses, the collagen organization dramatically changes, acquiring an orthogonal arrangement. Fourier transform analysis affirmed this observation and revealed that adjacent collagen lamellae display an angular displacement progressing from the epithelium layer towards the endothelium. These data indicate that the collagen organization of the wounded embryonic cornea recapitulate the native macrostructure.

Periocular neural crest cell differentiation into corneal endothelium is influenced by signals in the nascent corneal environment.

During ocular development, periocular neural crest cells (pNC) migrate into the region between the lens and presumptive corneal epithelium to form the corneal endothelium and stromal keratocytes. Although defects in neural crest cell development are associated with ocular dysgenesis, very little is known about the molecular mechanisms involved in this process. This study focuses on the corneal endothelium, a monolayer of specialized cells that are essential for maintaining normal hydration and transparency of the cornea. In avians, corneal endothelial cells are first to be specified from the pNC during their migration into the presumptive corneal region. To investigate the signals required for formation of the corneal endothelium, we utilized orthotopic and heterotopic injections of dissociated quail pNC into chick ocular regions. We find that pNC are multipotent and that the nascent cornea is competent to induce differentiation of ectopically injected pNC into corneal endothelium. Injected pNC downregulate expression of multipotency transcription factors and upregulate genes that are consistent with ontogenesis of the chick corneal endothelium. Importantly, we showed that TGFß2 is expressed by the nascent lens and the corneal endothelium, and that TGFß signaling plays a critical role in changing the molecular signature of pNC in vitro. Collectively, our results demonstrate the significance of the ocular environmental cues towards pNC differentiation, and have potential implications for clinical application of stem cells in the anterior segment.

Primary cilia deficiency in neural crest cells models anterior segment dysgenesis in mouse.

Defects affecting tissues of the anterior segment (AS) of the eye lead to a group of highly debilitating disorders called Anterior Segment Dysgenesis (ASD). Despite the identification of some causative genes, the pathogenesis of ASD remains unclear. Interestingly, several ciliopathies display conditions of the AS. Using conditional targeting of Ift88 with Wnt1-Cre, we show that primary cilia of neural crest cells (NCC), precursors of most AS structures, are indispensable for normal AS development and their ablation leads to ASD conditions including abnormal corneal dimensions, defective iridocorneal angle, reduced anterior chamber volume and corneal neovascularization. Mechanistically, NCC cilia ablation abolishes hedgehog (Hh) signaling in the periocular mesenchyme (POM) canonically activated by choroid-secreted Indian Hh, reduces proliferation of POM cells surrounding the retinal pigment epithelium and decreases the expression of Foxc1 and Pitx2, two transcription factors identified as major ASD causative genes. Thus, we uncovered a signaling axis linking cilia and ASD.

Cell-independent matrix configuration in early corneal development.

Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3.

Transcriptomic analysis of differential gene expression during chick periocular neural crest differentiation into corneal cells.

BACKGROUND: Multipotent neural crest cells (NCC) contribute to the corneal endothelium and keratocytes during ocular development, but the molecular mechanisms that underlie this process remain poorly understood. We performed RNA-Seq analysis on periocular neural crest (pNC), corneal endothelium, and keratocytes and validated expression of candidate genes by in situ hybridization. RESULTS: RNA-Seq profiling revealed enrichment of genes between pNC and neural crest-derived corneal cells, which correspond to pathways involved in focal adhesion, ECM-receptor interaction, cell adhesion, melanogenesis, and MAPK signaling. Comparisons of candidate NCC genes to ocular gene expression revealed that majority of the NCC genes are expressed in the pNC, but they are either differentially expressed or maintained during corneal development. Several genes involved in retinoic acid, transforming growth factor-ß, and Wnt signaling pathways and their modulators are also differentially expressed. We identified differentially expressed transcription factors as potential downstream candidates that may instruct expression of genes involved in establishing corneal endothelium and keratocyte identities. CONCLUSION: Combined, our data reveal novel changes in gene expression profiles as pNC differentiate into highly specialized corneal endothelial cells and keratocytes. These data serve as platform for further analyses of the molecular networks involved in NCC differentiation into corneal cells and provide insights into genes involved in corneal dysgenesis and adult diseases.

Expression of the heparin-binding growth factors Midkine and pleiotrophin during ocular development.

Midkine (MDK) and Pleiotrophin (PTN) belong to a group of heparin-binding growth factors that has been shown to have pleiotropic functions in various biological processes during development and disease. Development of the vertebrate eye is a multistep process that involves coordinated interactions between neuronal and non-neuronal cells, but very little is known about the potential function of MDK and PTN in these processes. In this study, we demonstrate by section in situ hybridization, the spatiotemporal expression of MDK and PTN during ocular development in chick and mouse. We show that MDK and PTN are expressed in dynamic patterns that overlap in a few non-neuronal tissues in the anterior eye and in neuronal cell layers of the posterior eye. We show that the expression patterns of MDK and PTN are only conserved in a few tissues in chick and mouse but they overlap with the expression of some of their receptors LRP1, RPTPZ, ALK, NOTCH2, ITGß1, SDC1, and SDC3. The dynamic expression patterns of MDK, PTN and their receptors suggest that they function together during the multistep process of ocular development and they may play important roles in cell proliferation, adhesion, and migration of neuronal and non-neuronal cells.

Transformation of the Transcriptomic Profile of Mouse Periocular Mesenchyme During Formation of the Embryonic Cornea.

Purpose: Defects in neural crest development are a major contributing factor in corneal dysgenesis, but little is known about the genetic landscape during corneal development. The purpose of this study was to provide a detailed transcriptome profile and evaluate changes in gene expression during mouse corneal development. Methods: RNA sequencing was used to uncover the transcriptomic profile of periocular mesenchyme (pNC) isolated at embryonic day (E) 10.5 and corneas isolated at E14.5 and E16.5. The spatiotemporal expression of several differentially expressed genes was validated by in situ hybridization. Results: Analysis of the whole-transcriptome profile between pNC and embryonic corneas identified 3815 unique differentially expressed genes. Pathway analysis revealed an enrichment of differentially expressed genes involved in signal transduction (retinoic acid, transforming growth factor-ß, and Wnt pathways) and transcriptional regulation. Conclusions: Our analyses, for the first time, identify a large number of differentially expressed genes during progressive stages of mouse corneal development. Our data provide a comprehensive transcriptomic profile of the developing cornea. Combined, these data serve as a valuable resource for the identification of novel regulatory networks crucial for the advancement of studies in congenital defects, stem cell therapy, bioengineering, and adult corneal diseases.

Knockdown of CXCL14 disrupts neurovascular patterning during ocular development.

The C-X-C motif ligand 14 (CXCL14) is a recently discovered chemokine that is highly conserved in vertebrates and expressed in various embryonic and adult tissues. CXCL14 signaling has been implicated to function as an antiangiogenic and anticancer agent in adults. However, its function during development is unknown. We previously identified novel expression of CXCL14 mRNA in various ocular tissues during development. Here, we show that CXCL14 protein is expressed in the anterior eye at a critical time during neurovascular development and in the retina during neurogenesis. We report that RCAS-mediated knockdown of CXCL14 causes severe neural defects in the eye including precocious and excessive innervation of the cornea and iris. Absence of CXCL14 results in the malformation of the neural retina and misprojection of the retinal ganglion neurons. The ocular neural defects may be due to loss of CXCL12 modulation since recombinant CXCL14 diminishes CXCL12-induced axon growth in vitro. Furthermore, we show that knockdown of CXCL14 causes neovascularization of the cornea. Altogether, our results show for the first time that CXCL14 plays a critical role in modulating neurogenesis and inhibiting ectopic vascularization of the cornea during ocular development.

PlexinD1 is required for proper patterning of the periocular vascular network and for the establishment of corneal avascularity during avian ocular development.

The anterior eye is comprised of an avascular cornea surrounded by a dense periocular vascular network and therefore serves as an excellent model for angiogenesis. Although signaling through PlexinD1 underlies various vascular patterning events during embryonic development, its role during the formation of the periocular vascular network is yet to be determined. Our recent study showed that PlexinD1 mRNA is expressed by periocular angioblasts and blood vessels during ocular vasculogenesis in patterns that suggest its involvement with Sema3 ligands that are concurrently expressed in the anterior eye. In this study, we used in vivo knockdown experiments to determine the role of PlexinD1 during vascular patterning in the anterior eye of the developing avian embryos. Knockdown of PlexinD1 in the anterior eye caused mispatterning of the vascular network in the presumptive iris, which was accompanied by lose of vascular integrity and profuse hemorrhaging in the anterior chamber. We also observed ectopic vascularization of the cornea in PlexinD1 knockdown eyes, which coincided with the formation of the limbal vasculature in controls. Finally we show that Sema3E and Sema3C transcripts are expressed in ocular tissue that is devoid of vasculature. These results indicate that PlexinD1 plays a critical role during vascular patterning in the iris and limbus, and is essential for the establishment of corneal avascularity during development. We conclude that PlexinD1 is involved in vascular response to antiangiogenic Sema3 signaling that guides the formation of the iris and limbal blood vessels by inhibiting VEGF signaling.

Corneal Development: Different Cells from a Common Progenitor.

Development of the vertebrate cornea is a multistep process that involves cellular interactions between various ectodermal-derived tissues. Bilateral interactions between the neural ectoderm-derived optic vesicles and the cranial ectoderm give rise to the presumptive corneal epithelium and other epithelia of the ocular surface. Interactions between the neural tube and the adjacent ectoderm give rise to the neural crest cells, a highly migratory and multipotent cell population. Neural crest cells migrate between the lens and presumptive corneal epithelium to form the corneal endothelium and the stromal keratocytes. The sensory nerves that abundantly innervate the corneal stroma and epithelium originate from the neural crest- and ectodermal placode-derived trigeminal ganglion. Concomitant with corneal innervation is the formation of the limbal vascular plexus and the establishment of corneal avascularity. This review summarizes historical and current research to provide an overview of the genesis of the cellular layers of the cornea, corneal innervation, and avascularity.

Quail-chick chimeras and eye development.

The quail-chick chimera is a stable and precise labeling technique that allows tracing of definite cells and their progeny without interfering with normal development of two related avian species. This technique utilizes the transplantation of quail tissues into chick embryo or vice versa. The region of interest (graft) is removed from the donor and replaced in the stage-matched host embryo. The quail-chick chimeras can be analyzed by immunolabeling donor-derived cells with species-specific antibodies, or by differential staining of the nucleus. The use of the quail-chick chimera technique is valuable to eye development studies since its formation involves coalition of tissues from different embryonic origins: the ectoderm, neuroectoderm, and neural crest cells derived from the interaction between the ectoderm and neural ectoderm. This chapter describes the protocols for using quail-chick chimeras to identify neural crest- and ectoderm-derived components of the eye. This technique can be used in combination with molecular biology techniques in functional studies to determine the cellular and tissue interactions involved in eye development.

Sema3A maintains corneal avascularity during development by inhibiting Vegf induced angioblast migration.

Corneal avascularity is important for optical clarity and normal vision. However, the molecular mechanisms that prevent angioblast migration and vascularization of the developing cornea are not clear. Previously we showed that periocular angioblasts and forming ocular blood vessels avoid the presumptive cornea despite dynamic ingression of neural crest cells. In the current study, we investigate the role of Semaphorin3A (Sema3A), a cell guidance chemorepellent, on angioblast migration and corneal avascularity during development. We show that Sema3A, Vegf, and Nrp1 are expressed in the anterior eye during cornea development. Sema3A mRNA transcripts are expressed at significantly higher levels than Vegf in the lens that is positioned adjacent to the presumptive cornea. Blockade of Sema3A signaling via lens removal or injection of a synthetic Sema3A inhibitor causes ectopic migration of angioblasts into the cornea and results in its subsequent vascularization. In addition, using bead implantation, we demonstrate that exogenous Sema3A protein inhibits Vegf-induced vascularization of the cornea. In agreement with these findings, loss of Sema/Nrp1 signaling in Nrp1(Sema-) mutant mice results in ectopic angioblasts and vascularization of the embryonic mouse corneas. Altogether, our results reveal Sema3A signaling as an important cue during the establishment of corneal avascularity in both chick and mouse embryos. Our study introduces cornea development as a new model for studying the mechanisms involved in vascular patterning during embryogenesis and it also provides new insights into therapeutic potential for Sema3A in neovascular diseases.

Wounded embryonic corneas exhibit nonfibrotic regeneration and complete innervation.

PURPOSE: Wound healing in adult corneas is characterized by activation of keratocytes and extracellular matrix (ECM) synthesis that results in fibrotic scar formation and loss of transparency. Since most fetal wounds heal without scaring, we investigated the regenerative potential of wounded embryonic corneas. METHODS: On embryonic day (E) 7 chick corneas were wounded by making a linear incision traversing the epithelium and anterior stroma. Wounded corneas were collected between E7 and E18, and analyzed for apoptosis, cell proliferation, staining of ECM components, and corneal innervation. RESULTS: Substantial wound retraction was observed within 16-hours postwounding (hpw) and partial re-epithelialized by 5-days postwounding (dpw). Corneal wounds were fully re-epithelialized by 11 dpw with no visible scars. There was no difference in the number of cells undergoing apoptosis between wounded and control corneas. Cell proliferation was reduced in the wounded corneas, albeit mitotic cells in the regenerating epithelium. Staining for alpha-smooth muscle actin (α-SMA), tenascin, and fibronectin was vivid but transient at the wound site. Staining for procollagen I, perlecan, and keratan sulfate proteoglycan was reduced at the wound site. Wounded corneas were fully regenerated by 11 dpw and showed similar patterns of staining for ECM components, albeit an increase in perlecan staining. Corneal innervation was inhibited during wound healing, but regenerated corneas were innervated similar to controls. CONCLUSIONS: These data show that minimal keratocyte activation, rapid ECM reconstruction, and proper innervation occur during nonfibrotic regeneration of the embryonic cornea.

Expression of CXCL12 and CXCL14 during eye development in chick and mouse.

Vertebrate eye development is a complex multistep process coordinated by signals from the lens, optic cup and periocular mesenchyme. Although chemokines are increasingly being recognized as key players in cell migration, proliferation, and differentiation during embryonic development, their potential role during eye development has not been examined. In this study, we demonstrate by section in situ hybridization that CXCL12 and CXCL14 are expressed during ocular development. CXCL12 is expressed in the periocular mesenchyme, ocular blood vessels, retina, and eyelid mesenchyme, and its expression pattern is conserved between chick and mouse in most tissues. Expression of CXCL14 is localized in the ocular ectoderm, limbal epithelium, scleral papillae, eyelid mesenchyme, corneal keratocytes, hair follicles, and retina, and it was only conserved in the upper eyelid ectoderm of chick and mouse. The unique and non-overlapping patterns of CXCL12 and CXCL14 expression in ocular tissues suggest that these two chemokines may interact and have important functions in cell proliferation, differentiation and migration during eye development.