R-3 Repository :: Browsing by Author "Lwigale, Peter Y."

Browsing by Author "Lwigale, Peter Y."

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A Technique to Increase Accessibility to Late-Stage Chick Embryos for In Ovo Manipulations
(Wiley, 2013) Spurlin, James III; Lwigale, Peter Y.
Background: During early development, avian embryos are easily accessible in ovo for transplantations and experimental perturbations. However, these qualities of the avian embryonic model rapidly wane shortly after embryonic day (E)4 when the embryo is obscured by extraembryonic membranes, making it difficult to study developmental events that occur at later stages in vivo. Results: In this study, we describe a multistep method that involves initially windowing eggs at E3, followed by dissecting away extraembryonic membranes at E5 to facilitate embryo accessibility in ovo until later stages of development. The majority of the embryos subjected to this technique remain exposed between E5 and E8, then become gradually displaced by the growing allantois from posterior to anterior regions. Conclusions: Exposed embryos are viable and compatible with embryological and modern developmental biology techniques including tissue grafting and ablation, gene manipulation by electroporation, and protein expression. This technique opens up new avenues for studying complex cellular interactions during organogenesis and can be further extrapolated to regeneration and stem cell studies.
BMP3 inhibits TGFβ2-mediated myofibroblast differentiation during wound healing of the embryonic cornea
(Springer Nature, 2022) Spurlin, James W.; Garis, Matthew R.; Lwigale, Peter Y.
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 TGFβ2 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 TGFβ2-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.
Cell-independent matrix configuration in early corneal development
(Elsevier, 2019) Young, Robert D.; Knupp, Carlo; Koudouna, Elena; Ralphs, James R.; Ma, Yanhui; Lwigale, Peter Y.; Jester, James V.; Quantock, Andrew J.
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.
Distinct Roles for Neuropilin1 and Neuropilin2 during Mouse Corneal Innervation
(Public Library of Science, 2012) McKenna, Chelsey C.; Munjaal, Ravi P.; Lwigale, Peter Y.
Trigeminal sensory innervation of the cornea is critical for protection and synthesis of neuropeptides required for normal vision. Little is known about axon guidance during mammalian corneal innervation. In contrast to the chick where a pericorneal nerve ring forms via Npn/Sema signaling, mouse corneal axons project directly into the presumptive cornea without initial formation of an analogous nerve ring. Here we show that during development of the mouse cornea, Npn1 is strongly expressed by the trigeminal ganglion whereas Npn2 is expressed at low levels. At the same time Sema3A and Sema3F are expressed in distinct patterns in the ocular tissues. Npn1sema−/− mutant corneas become precociously and aberrantly innervated by nerve bundles that project further into the corneal stroma. In contrast, stromal innervation was not affected in Npn2−/− mutants. The corneal epithelium was prematurely innervated in both Npn1sema−/− and Npn2−/− mutants. These defects were exacerbated in Npn1sema−/−;Npn2−/− double mutants, which in addition showed ectopic innervation of the region between the optic cup and lens vesicle. Collectively, our data show that Sema3A/Npn1 and Sema3F/Npn2 signaling play distinct roles and both are required for proper innervation of the mouse cornea.
Expression of CXCL12 and CXCL14 during eye development in chick and mouse
(Elsevier, 2013) Ojeda, Ana F.; Munjaal, Ravi P.; Lwigale, Peter Y.
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.
Expression of pro- and anti-angiogenic factors during the formation of the periocular vasculature and development of the avian cornea
(John Wiley & Sons, Inc., 2013) Kwiatkowski, Sam; Munjaal, Ravi P.; Lee, Teresa; Lwigale, Peter Y.
Background: During embryonic development, endothelial precursor cells (angioblasts) migrate relatively long distances to form the primary vascular plexus. The migratory behavior of angioblasts and localization of the primitive blood vessels is tightly regulated by pro-angiogenic and anti-angiogenic factors encountered in the embryonic environment. Despite the importance of corneal avascularity to proper vision, it is not known when avascularity is established in the developing cornea and how pro- and anti-angiogenic factors regulate this process. Results and Discussion: Using Tg(tie1:H2B:eYFP) transgenic quail embryos to visualize fluorescently labeled angioblasts, we show that the presumptive cornea remains avascular despite the invasion of cells from the periocular region where migratory angioblasts reside and form the primary vasculature. Semiquantitative reverse transcriptase polymerase chain reaction analysis and spatiotemporal examination of gene expression revealed that pro- and anti-angiogenic factors were expressed in patterns indicating their potential roles in angioblast guidance. Conclusions: Our findings show for the first time that chick corneal avascularity is established and maintained during development as the periocular vasculature forms. We also identify potential candidate pro- and anti-angiogenic factors that may play crucial roles during vascular patterning in the anterior eye.
Human Fetal Keratocytes Have Multipotent Characteristics in the Developing Avian Embryo
(Mary Ann Liebert, Inc., 2013) Chao, Jennifer R.; Bronner, Marianne E.; Lwigale, Peter Y.
The human cornea contains stem cells that can be induced to express markers consistent with multipotency in cell culture; however, there have been no studies demonstrating that human corneal keratocytes are multipotent. The objective of this study is to examine the potential of human fetal keratocytes (HFKs) to differentiate into neural crest-derived tissues when challenged in an embryonic environment. HFKs were injected bilaterally into the cranial mesenchyme adjacent to the neural tube and the periocular mesenchyme in chick embryos at embryonic days 1.5 and 3, respectively. The injected keratocytes were detected by immunofluorescence using the human cell-specific marker, HuNu. HuNu-positive keratocytes injected along the neural crest pathway were localized adjacent to HNK-1-positive migratory host neural crest cells and in the cardiac cushion mesenchyme. The HuNu-positive cells transformed into neural crest derivatives such as smooth muscle in cranial blood vessels, stromal keratocytes, and corneal endothelium. However, they failed to form neurons despite their presence in the condensing trigeminal ganglion. These results show that HFKs retain the ability to differentiate into some neural crest-derived tissues. Their ability to respond to embryonic cues and generate corneal endothelium and stromal keratocytes provides a basis for understanding the feasibility of creating specialized cells for possible use in regenerative medicine.
Mechanisms of Corneal Development and Wound Healing
(2020-03-19) Babushkina, Anna; Lwigale, Peter Y.
Deep corneal injuries are one of the major causes of vision impairment and blindness. The shortage in available donor corneas has prompted researchers to look into developing alternative means of treating corneal injuries. Previously, our lab demonstrated that embryonic cornea heals scar-free. The purpose of this thesis work is to elucidate molecular pathways and signals during corneal development and to understand how the embryonic cornea heals. My current research uses the chick as a model organism to study embryonic cornea. 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 multiple ocular dysgeneses are associated with defects in neural crest cell development, very little is known about the molecular mechanisms behind the process. To investigate the role of the environment in formation of the corneal endothelium and the stroma, I introduce a model for studying pNC development in the ocular environment. I demonstrate that upon orthotopic injection of pNC into the periocular region the cells timely migrate and differentiate into multiple ocular derivatives confirming that pNC are multipotent. Using this model, I show that the presumptive cornea is conducive to endothelial differentiation between embryonic day (E) 3-E7. At the same time, the periocular region does not induce corneal migration of the heterotopically injected pNC at E5 suggesting environmental changes in favor of an alternative ocular fate. I further demonstrate that corneal stroma at E7 may have the potential to induce pNC-to-keratocyte differentiation on the spot. To investigate specific signals required for pNC differentiation I focus on the corneal endothelium, which is the first corneal layer to be specified from the pNC in both avians,and humans. I find that the nascent cornea is competent to induce differentiation of ectopically injected pNC into corneal endothelium. Injected pNC downregulate the expression of multipotency transcription factors and upregulate endothelium-related genes. I show that TGFβ2 signaling in the nascent corneal environment plays a critical role in changing the molecular signature of pNC during the formation of the corneal endothelium. Additionally, proper arrangement of the collagen and the proteoglycans in the stromal extracellular matrix (ECM) is essential for functional transparency and light refraction of the cornea. Here, I present that the cornea is able to recapitulate its three-dimensional collagen structure during wound healing. The results of the presented work shed light on the complex molecular network involved in pNC differentiation in the eye and on the process of embryonic wound healing. Together, these findings could aid the development of alternative treatments for corneal injuries and disorders.
Recapitulation of normal collagen architecture in embryonic wounded corneas
(Springer Nature, 2020) Koudouna, Elena; Spurlin, James W.; Babushkina, Anna; Quantock, Andrew J.; Jester, James V.; Lwigale, Peter Y.
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.
Transformation of the Transcriptomic Profile of Mouse Periocular Mesenchyme During Formation of the Embryonic Cornea
(ARVO, 2019) Ma, Justin; Lwigale, Peter Y.
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-β, 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.

Browsing by Author "Lwigale, Peter Y."

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