Browsing by Author "Spurlin, James W."

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    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.
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    Evolution of the ﾠPerlecan/HSPG2ﾠ Gene and Its Activation in Regeneratingﾠ Nematostella vectensis
    (Public Library of Science, 2015) Warren, Curtis R.; Kassir, Elias; Spurlin, James W.; Martinez, Jerahme R.; Putnam, Nicholas H.; Farach-Carson, Mary C.
    The heparan sulfate proteoglycan 2 (HSPG2)/perlecan gene is ancient and conserved in all triploblastic species. Its presence maintains critical cell boundaries in tissue and its large (up to ~900 kDa) modular structure has prompted speculation about the evolutionary origin of the gene. The gene’s conservation amongst basal metazoans is unclear. After the recent sequencing of their genomes, the cnidarian Nematostella vectensis and the placozoanTrichoplax adhaerens have become favorite models for studying tissue regeneration and the evolution of multicellularity. More ancient basal metazoan phyla include the poriferan and ctenophore, whose evolutionary relationship has been clarified recently. Our in silico and PCR-based methods indicate that the HSPG2 gene is conserved in both the placozoan and cnidarian genomes, but not in those of the ctenophores and only partly in poriferan genomes.HSPG2 also is absent from published ctenophore and Capsaspora owczarzaki genomes. The gene in T. adhaerens is encoded as two separate but genetically juxtaposed genes that house all of the constituent pieces of the mammalian HSPG2 gene in tandem. These genetic constituents are found in isolated genes of various poriferan species, indicating a possible intronic recombinatory mechanism for assembly of the HSPG2 gene. Perlecan’s expression during wound healing and boundary formation is conserved, as expression of the gene was activated during tissue regeneration and reformation of the basement membrane of N.vectensis. These data indicate that the complex HSPG2 gene evolved concurrently in a common ancestor of placozoans, cnidarians and bilaterians, likely along with the development of differentiated cell types separated by acellular matrices, and is activated to reestablish these tissue borders during wound healing.
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    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.
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    Visualization of RNA virus infection in a marine protist with a universal biomarker
    (Springer Nature, 2023) Coy, Samantha R.; Utama, Budi; Spurlin, James W.; Kim, Julia G.; Deshmukh, Harshavardhan; Lwigale, Peter; Nagasaki, Keizo; Correa, Adrienne M. S.
    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.