Quantification of Dynamic Epithelial Sheet Architecture in Botryllus Schlosseri Using 2-D & 3-D Image Analysis

<p>Epithelial tubules form critical structures in various body tissues; how- ever, since they are difficult to access experimentally, their architecture and dynamics are not well understood. Here we examine the dynamic remodeling of epithelial tubes in vivo using a novel and uniquely accessible model system: the extracorporeal vasculature of <em>Botryllus schlosseri</em> (sea squirt). In <em>Botryllus</em>, massive retraction of blood vessels can be triggered without loss of barrier function, through (i) disrupting collagen crosslinking in the basement membrane using β-aminopropionitrile (BAPN); or (ii) disrupting the integrin pathway through inhibition of focal adhesion kinase (FAK). We performed stereographic projections of 3-dimensional high-resolution confocal scans to generate an `unwrapped' 2-dimensional map of the cylindrical blood vessels, on which we performed quantitative analysis of epithelial morphology. In normal vessels, we found the cells to be planar polarized with curvature - dependent axial elongation of cells, and a robust circumferential alignment of actin bundles. While we found no measurable differences in morphology between normal and BAPN treated vessels, FAK-inhibited vessels have significantly smaller cells with predominant circumferential cell orientation and a loss of actin bundles. Our results demonstrate the feasibility of <em>Botryllus schlosseri</em> for imaging-based studies of dynamic epithelial remodeling. They also suggest a critical role of integrin in the maintenance of epithelial morphology.</p> <p>In a second on going study, we are analyzing the 3-dimensional confocal scans of live vasculature. We were able to successfully produce the stereographic projections of the live blood vessels under control, BAPN treated and FAK inhibitor treated conditions and are working on extracting the morphological properties. We hope to identify and quantify the multiple mechanisms used by regressing <em>Botryllus</em> vasculature.</p> <p>In a third on going study, we are studying the blood ow rates and the branching behaviors of blood vessels in old and young <em>Botryllus schlosseri</em>. While slowing of blood ow and narrowing of blood vessels was visually shown to be the first stage of aging in seasquirts, no quantification has been done about the same. We find that blood vessels significantly narrow down in older animals and are working on quantifying the amount of branching in old and young animals.</p> <p>In a fourth and final study, the role of membrane trafficking protein Rab35 on interface shortening during germband extension in <em>Drosophila melanogaster</em> was studied. Germband extension (GBE) is a developmental process that drives nonuniform tissue elongation through oriented cell intercalation. During GBE, interfaces along the dorsal-ventral (DV) axis shrink and new interfaces are formed along the AP axis. The results of this study showed that Rab35 compartments are enriched at the actively contracting interfaces and the disruption of Rab35 lead to failure of interface shortening.</p> <p>As each of these projects is different from one another thus making it difficult to form a single narrative, I only talk about the first study in this thesis.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Year dc:date.available

2020

Author dc:creator

• Madhu, Roopa

Contributors dc:contributor

• Dinah Loerke
• Jing Li
• J. Todd Blankenship
• Joseph K. Angleson
• Schuyler Van Engelenburg

• Botryllus schlosseri
• Drosophila melanogaster
• Biochemistry, Biophysics, and Structural Biology
• Biological and Chemical Physics
• Biophysics
• Cell Biology

Statement dc:rights

• <p>Copyright is held by the author. User is responsible for all copyright compliance.</p>

Language dc:language

en

Repository record dc:identifier

https://digitalcommons.du.edu/etd/1799

OAI identifier oai:identifier

oai:digitalcommons.du.edu:etd-2791