CHARACTERIZATION OF NOVEL MOLECULAR TOOLS AND THEIR APPLICATION TO STUDIES OF HEART DEVELOPMENT

Ph.DDOCTOR OF PHILOSOPH

Optogenetic in vivo cell manipulation in KillerRed-expressing zebrafish transgenics

<p>Abstract</p> <p>Background</p> <p>KillerRed (KR) is a novel photosensitizer that efficiently generates reactive oxygen species (ROS) in KR-expressing cells upon intense green or white light illumination <it>in vitro</it>, resulting in damage to their plasma membrane and cell death.</p> <p>Results</p> <p>We report an <it>in vivo </it>modification of this technique using a fluorescent microscope and membrane-tagged KR (mem-KR)-expressing transgenic zebrafish. We generated several stable zebrafish <it>Tol2 </it>transposon-mediated enhancer-trap (ET) transgenic lines expressing mem-KR (SqKR series), and mapped the transposon insertion sites. As mem-KR accumulates on the cell membrane and/or Golgi, it highlights cell bodies and extensions, and reveals details of cellular morphology. The photodynamic property of KR made it possible to damage cells expressing this protein in a dose-dependent manner. As a proof-of-principle, two zebrafish transgenic lines were used to affect cell viability and function: SqKR2 expresses mem-KR in the hindbrain rhombomeres 3 and 5, and elsewhere; SqKR15 expresses mem-KR in the heart and elsewhere. Photobleaching of KR by intense light in the heart of SqKR15 embryos at lower levels caused a reduction in pumping efficiency of the heart and pericardial edema and at higher levels - in cell death in the hindbrain of SqKR2 and in the heart of SqKR15 embryos.</p> <p>Conclusions</p> <p>An intense illumination of tissues expressing mem-KR affects cell viability and function in living zebrafish embryos. Hence, the zebrafish transgenics expressing mem-KR in a tissue-specific manner are useful tools for studying the biological effects of ROS.</p

Collective Cell Migration Drives Morphogenesis of the Kidney Nephron

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis

Synthesis and antibacterial study of sulfobetaine/quaternary ammonium-modified star-shaped poly[2-(dimethylamino)ethyl methacrylate]-based copolymers with an inorganic core

Star polymers with poly[2-(dimethylamino)ethyl methacrylate] as the arms and POSS as the core (POSS-g-PDMA) were synthesized by atom transfer radical polymerization (ATRP). The effect of molecular weight on the antibacterial activity was studied and lower molecular weight POSS-g-PDMA has better bactericidal activity as measured by the minimum inhibition concentration. POSS-g-PDMA was further modified by various techniques to increase hydrophilicity in attempting to improve their antifouling activity without compromising bactericidal activity. POSS-g-PDMA was quaternized to different degrees and the antibacterial activities of the obtained quaternary polymers were studied; the antibacterial activity decreased as the degree of quaternization increased. Finally, cationic-zwitterionic polymers with both random and block structures, where PDMA and poly(sulfobetaine) were cationic and zwitterionic blocks respectively, were synthesized. The random cationic-zwitterionic polymers showed poor antibacterial activity while the block polymers retained the antibacterial activity of the pristine POSS-g-PDMA. The block copolymers of POSS-g-(PDMA-b-polysulfobetaine) showed enhanced antifouling property and serum stability as seen by their nanoparticle size stability in the presence of serum and reduced red blood cell aggregation. The antibacterial kinetics showed that E. coli can be killed within 30 min by both random and block polymers. Finally, block polymers showed low toxicity to zebrafish embryo and could be potentially used in aquaculture antibacterial applications.MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore)Accepted versio

Synthesis and Antibacterial Study of Sulfobetaine/Quaternary Ammonium-Modified Star-Shaped Poly[2-(dimethylamino)ethyl methacrylate]-Based Copolymers with an Inorganic Core

Cationic polymethacrylates are interesting candidates for bacterial disinfectants since they can be made in large-scale by various well-established polymerization techniques such as atom transfer radical polymerization (ATRP). However, they are usually toxic or ineffective in serum and various strategies to improve their biocompatibility or nonfouling property have often resulted in compromised bactericidal activity. Also, star-shaped polymers are less explored than linear polymers for application as antibacterial compounds. In this paper, star polymers with poly­[2-(dimethylamino)­ethyl methacrylate] (PDMA) as the arms and polyhedral oligomeric silsesquioxane (POSS) as the core (POSS-<i>g</i>-PDMA) were successfully synthesized by ATRP. The minimum inhibition concentrations (MICs) of the synthesized POSS-<i>g</i>-PDMA are in the range of 10–20 μg/mL. POSS-<i>g</i>-PDMA was further modified by various hydrophilization strategies in attempting to reduce hemolysis. With quaternization of POSS-<i>g</i>-PDMA, the antibacterial activities of the obtained quaternary polymers are almost unchanged and the copolymers become relatively nonhemolytic. We also copolymerized sulfobetaine (SB) with POSS-<i>g</i>-PDMA to obtain random and block PDMA-<i>co</i>-PSB arm structures, where the PDMA and poly­(sulfobetaine) were the cationic and zwitterionic blocks, respectively. The random cationic–zwitterionic POSS-<i>g</i>-(PDMA-<i>r</i>-PSB) copolymers showed poor antibacterial activity, while the block POSS-<i>g</i>-(PDMA-<i>b</i>-PSB) copolymers retained the antibacterial and hemolytic activity of the pristine POSS-<i>g</i>-PDMA. Further, the block copolymers of POSS-<i>g</i>-(PDMA-<i>b</i>-PSB) showed enhanced antifouling property and serum stability as seen by their nanoparticle size stability in the presence of serum and reduced red blood cell aggregation; the POSS-<i>g</i>-(PDMA-<i>b</i>-PSB) also somewhat retained its MIC in blood unlike the quaternized or random zwitterionic copolymers. The antibacterial kinetics study showed that Escherichia coli can be killed within 30 min by both random and block copolymers of POSS-<i>g</i>-(PDMA-<i>co</i>-PSB). Finally, our POSS star polymers showed low toxicity to zebrafish embryo and could be potentially used in aquaculture antibacterial applications