eprintid: 10100274 rev_number: 20 eprint_status: archive userid: 608 dir: disk0/10/10/02/74 datestamp: 2020-09-10 10:35:07 lastmod: 2021-07-01 06:10:21 status_changed: 2020-09-10 10:35:07 type: thesis metadata_visibility: show creators_name: Stapornwongkul, Kristina Sukanya title: Engineering a Diffusion-Based Signalling Gradient in vivo ispublished: unpub divisions: UCL divisions: A01 divisions: B02 divisions: C08 divisions: D09 note: Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. abstract: Morphogens are secreted signalling molecules that disperse from their region of production to form concentration gradients in the adjacent tissue. By inducing cellular responses in a concentration-dependent manner, morphogens organise pattern formation in various tissues. How extracellular protein gradients form has remained controversial, especially in epithelia. In this work, I used a forward-engineering approach to assess the minimal requirements for diffusion-based gradient formation by transforming the otherwise inert green fluorescent protein (GFP) into an in vivo morphogen. I found that, on its own, GFP cannot form a detectable gradient in the Drosophila wing disc, the epithelial pouch that gives rise to the adult wing. However, GFP does form a gradient in the presence of binding partners engineered from anti-GFP nanobodies. The gradient can be formally described by a diffusion-degradation model that considers GFP leakage into the hemolymph, the larval blood, and GFP reentering into the tissue. To test whether GFP gradients can faithfully provide positional information, I substituted GFP for Dpp, a bona fide morphogen, in the presence of Dpp receptors engineered to be responsive to GFP. GFP was able to partially rescue growth and patterning of the wing disc. However, GFP reentering the tissue from the hemolymph seemed to impair the patterning performance of the gradient. While increasing receptor number improved GFP retention, it also reduced the signalling range of the gradient. I found that the coexpression of low-affinity binding partners mimicking extracellular matrix components was able to both increase the gradient range and provide sufficient ligand retention. My results suggest that extracellular protein gradients can form by diffusion in epithelial tissues and that in a system where signalling receptors are the only binding partners, ligand leakage and signalling range are opposing constraints that can, however, be overcome by the coexpression of low-affinity binding partners. date: 2020-06-28 date_type: published oa_status: green full_text_type: other thesis_class: doctoral_open thesis_award: Ph.D language: eng thesis_view: UCL_Thesis primo: open primo_central: open_green verified: verified_manual elements_id: 1788609 lyricists_name: Stapornwongkul, Kristina lyricists_id: KSSTA33 actors_name: Stapornwongkul, Kristina actors_id: KSSTA33 actors_role: owner full_text_status: public pagerange: 1-184 pages: 184 event_title: UCL (University College London) institution: UCL (University College London) department: Division of Biosciences thesis_type: Doctoral citation: Stapornwongkul, Kristina Sukanya; (2020) Engineering a Diffusion-Based Signalling Gradient in vivo. Doctoral thesis (Ph.D), UCL (University College London). Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10100274/1/PhDThesis_K.Stapornwongkul_corrected.pdf