• ANALYSIS OF TEMPERATURE SENSITIVE CYK-1 MUTATIONS EFFECT ON MUSCLE DEVELOPMENT ON CAENORHABDITIS ELEGAN SLARVAE

      Pruyne, David; Laszlo, Arianna (2017)
      Sarcomeres are the most basic unit of muscle cells. Formins are actin regulatory proteins that are important for actin filament polymerization and nucleation, and might be responsible for the actin filamentassembly in sarcomeres. In Caenorhabditis elegans, two formins (CYK-1 and FHOD-1) were found in the body wall muscle (BWM), specifically in the Z-line of sarcomeres. Previously, BWM were analyzed in null cyk-1(ok2300) mutant worms, derived from heterozygous parents, and fhod-1(tm2363) mutantworms, andwere found to have smaller muscles than wild-type worms. Yet, there was still functional CYK-1 present in the worms due to inherited maternal CYK-1. To eliminate this potential source of CYK-1, cyk-1(or596ts) temperature sensitive mutant worms were used to allow for all CYK-1 to be non-functional at 26°C. The focus of this study was to understand the importance of cyk-1on muscle development in C. eleganslarvae. Wild-type, fhod-1(tm2363), cyk-1(or596ts), and fhod-1(tm2363);cyk-1(or596ts) double mutant wormsat L1 larval stage were observed for worm shape and muscle abnormalities. Abnormal worm shapes were observed in fhod-1, cyk-1, and more commonly in fhod-1;cyk-1mutant worms at permissive and restrictive temperatures. Abnormal muscle was observed in both permissive and restrictive temperatures forfhod-1, cyk-1, and fhod-1;cyk-1mutant worms that had abnormal worm shapes, while all worms that had normal body shape usually had normal muscle at both temperatures. Worms were alsotested to determine long-termeffects of cyk-1and fhod-1mutations on muscle development. Worms were held at permissive or restrictive temperatures for various times. Fhod-1and cyk-1mutant worms showed reduced muscle size compared to wild-type, while fhod-1;cyk-1mutant worms displayed a more severelyreduced muscle size. Next, cyk-1orfhod-1were tested to see whether they can rescue muscle cell size after mosaic expressionin a cyk-1(-) or fhod-1(-) mutant worm background. Fhod-1(+) muscle cells showeda significant increase in muscle cell size compared to fhod-1(-) cells, while cyk-1(+) muscle cells showeda non-significant increase in muscle size compared to cyk-1(+/-) cells. This shows cell-autonomousexpressionoffhod-1influencesmuscle growth, while cyk-1expression in another organ might influence muscle development.
    • ROLES FOR NETRIN-4 AND LAMININS CONTAINING THE β2 OR γ3 SUBUNITS IN CORNEAL DEVELOPMENT AND WOUND HEALING

      Brunken, William; Martino, Jeremiah (2016)
      Corneal diseases are among the top preventable causes of vision loss in the developed world. Injuries, infections, age-related diseases and congenital diseases are typically accompanied by defects in the extracellular matrix (ECM) or its remodeling. Laminins and laminin-related molecules, netrins, are ECM proteins that play an important yet not fully characterized role in corneal development, disease and wound recovery. With regulatory roles in cell proliferation, migration and neural guidance, the main research question addressed here is how netrin-4 and specific laminin subunits, i.e. the laminin β2 and γ3 chains, are involved in corneal development and wound recovery. A reverse genetics approach was employed to characterize the corneal phenotype in mice lacking the Ntn4, Lamb2, or Lamc3genes during development and wound healing.Immunohistochemistry data showed that netrin-4 and the laminin β2 and γ3 chains are components of corneal basement membranes. Genetic ablation of Lamb2, Lamc3or both genes led to hypo-proliferation of epithelial progenitors and hypo-innervation of the corneal surface, indicating regulatory roles in corneal development and possibly wound recovery. In addition to these effects at the surface, in the deep cornea, Descemet’s Membrane (DM) was thinner and endothelial cells displayed disruptions in tight-junction formation, likely resulting from an altered molecular composition of DM.On the other hand, deletion of Ntn4led to hyper-innervation of the corneal surface during development. When further characterizedin the context of wound recovery, adult Ntn4-/-mice showed a slightlyearlier reepithelialization, upregulated cell proliferation and reinnervation of wound area compared to wild-type littermates. Altogether, these data indicate that netrin-4 is likely a negative regulator of proliferative events in the cornea.Hownetrin-4 and laminin heterotrimers containing the laminin β2or γ3 subunits affect nerve-cell interactions in the cornea merits further inquiry. Given the observations presented herein, these molecules are important for corneal maintenance and have applications potential in strategies aimed toward treatment of corneal injuries, such as development of fully characterized, synthetic pro-regenerative membranes, or incorporation into implantable biomimetic scaffolds which can also mitigate current donor tissue shortages.
    • STUDYING RETINA DEVELOPMENT USING SMALL MOLECULE BMP INHIBITORS AND MAKING STABLE MOUSE EMBRYONIC STEM CELL LINES

      Viczian, Andrea; Keshvani, Caezaan (2013)
      Age related macular degeneration results in loss ofconephotoreceptors. Studies have not been able to efficientlytransplant cone cells, possiblydue to their limited numbersand lack of information about their development in the mammalian retina.Our lab has discoveredthat BMP signal inhibition is important for eye developmentin the frogas well as generating retinal cone photoreceptorsin mouse embryonic stem cell cultures. The frog, Xenopus laevis,can grow eyeswithin a few days; it is also amenable to transplantation and genetic alteration. In this study, two small molecule BMP inhibitors, LDN193189 and Dorsomorphin, causedshortened tails(dorsalized) phenotypein Xenopusembryos as well asexpansionofthe neural plate in neural stage embryos. This suggests that these chemical inhibitors can be used in place of the standard BMP antagonist, Noggin, in cell culture experiments. Mouse embryonic stem cell cultures treated with Noggin have been used to generate retinal progenitors that generate cone photoreceptorsin our lab. In order to further study cones indetail,we needed to enrich the population of cone photoreceptors from retinal progenitors by marking these cells in-­‐vitro. We generated stable mouse embryonic stem cells expressing a promoter that drives expression in rod/cone progenitor cells upstream from the mCherry fluorescent protein. This construct also contained aubiquitous promoterdriving an antibiotic resistance genefor antibiotic selection. In future studies, these stable ES celllines could be used to differentiate into rod/cone progenitors selected specifically by FAC sorting. Small molecule BMP inhibitors could be used in place of Noggin and analyzed in the generation of conecells.